- Email: [email protected]
Fibroblast growth factor receptors: lessons from the genes
REVIEWS
TIBS 2 3 - FEBRUARY1998
Fbrob]ast growth factor recepbrs: le,,sons from the genes Dav)d Burke, David Wilkes, Tom L. Blundetl and Sue Malcolm The fibroblast growth factor receptors (FGFRs) are a family of transmembrahe tyrosine kinases involved in signalling via interactions with the family of fibroblast growth factors (FGFs). Genetic findings have provided a way of dissecting these interactions. Mutations in three members of the FGFR family have been found in patients with birth defects involving craniosynostosis (premature fusion of the cranial sutures) or skeletal abnormalites. Analyses of the spectrum of mutations found predict that many of them will result in ligand-independent activation of the receptors. Amino acids have also been identified that are likely to be important in determining the specificity of FGFR-FGF interactions. VARIOUS METHODS have proved popular in attempts to translate biochemical findings about gene products into an understanding of the function of these genes in different tissues or the whole organism. Traditionally, mice with the gene knocked-out have been created and Drosophila melanogastermutants identified. In these studies, the gene in question is often rendered inactive, but in human developmental disorders the complete removal of the gene product arising from recessive or null mutations are the exception, rather than the rule. Gene function is often altered by a dosage effect, involving the loss of one copy (haploinsufficiency), or altered activity (gainof-function) as described here. Naturally occurring mutations provide an exceptional opportunity to study the different consequences of altered genes.
and survival, and have been implicated in pathological processes including anglogenesis, wound healing and cancer. The FGFRs interact with fibroblast growth factors (FGFs), of which at least 13 members have been characterized to date. Consequently, the interactions are extremely complex and difficult to disentangle by biochemical techniques ~. The FGF ligands interact with the extracellular portion of FGFR molecules, which consists of three immunoglobulin (lg)-like domains with short linking segments (Fig. 1). In the segment between Igl and lgll there is a run of the acidic residues g!utamic acid and aspartic acid,
(a)
D. Burke and T. Blundell are at the Department of Biochemistry, Universityof Cambridge, Tennis Court Road, Cambridge,UK CB2 1QW. D. Wilkes and S. Malcolm are at the Molecular Genetics Unit, Institute of Child Health, 30 Guilford Street, London, UK WC1N 1EH. Email: [email protected]
Igll
r~utations associated with birth defects A number of birth defects are associated with mutations in FGFRs Gable 1) which often involve incorrect fusion of the skull sutures or skeletal abnormalities. Box i describes the characteristics of the syndromes listed in Table I. Although uumerous different mutations haw been observed (over 25 in Crouzon syndrome alone), there is a distinct clustering in terms of type of mutation and position within the FGFR molecules (.see Ref. 4). One of the most striking features is the extent to which homologous
Iglll TM
ND The FGFRfamily The FGFRfamily consists of four closely related members, whose amino acid sequences are highly conserved between different members of the family and throughout evolution. They regulate a multitude of cellular processes, including cell growth, differentiation, migration
Igl
which is referred to as the 'acid box', as well as a three-amino acid motif hisfidinealanine-valine (HAV). The HAV sequence is also found in the cell-adhesion molecules N-CAM, L-CAM and N-cadherin, and it is thought to be involved in binding between molecules. There is a single transmembrane portion and, inside the cell, there is a split tyrosine-kinase domain. The overall complexity is increased by the existence of additional forms of the receptor, created by alternative splicing of mRNA. These receptors are either missing the first Ig domain (lgl), missing both Igl and the acid-box region or, more importantly, possess an alternative sequence for the C-terminal half of the third ]g-like domain encoded by a separate exon ~ (shaded black in Fig. la). Interaction between growth factor, receptor and heparan sulphate proteoglycans is required for activation of signalling. After ligand binding, receptor molecules dimerize and this is followed by autophosphorylation of several tyrosine residues in the intracelluar domain 3.
C-s-s-C--~
sP
Acid box
C-s-s-C
TK1-TK2
C-,.s-s-C
/\
j Pro253Arg
Ser372Cys Tyr375Cys
Gly384Arg
(b) FGFR2-111b
314 H S G I N S S - - N A E V L A L F ~ r T E A D A G E Y I C K V S N Y I G Q A N Q S A W L T V L P
359
FGFR2-111c
314 A A ~ ; V N T T D K E I E V L Y I R N V T F E D A G E Y T C L A G N S I G I S F H S A W L T V L P
361
Figure 1
(a) The fibroblast growth factor receptor, consisting of: the N-terminal signal peptide (SP); the acid-box region (a run of acidic residues); the three imrnunoglobulin-likedomains (Igl, Igll and Iglll); the transmernbrane region (TM); and the split bjrosine-kinase domain (TK1-TK2). The two cysteines forming the disulphide bridge are shown in each Ig-like domain. The dark region in Iglll is explained in the text. (b) Sequencealignmentof the alternative forms of the C-terminalhalf of the Igll[ domain (Iglllb and Iglllc) of FGFR2.The sequences shown represent the complete exon. Identical residues are shown in purple.
Copyright © 1998,ElsevierScience Ltd. All rights reserved. 0968-0004/98/$19.00 PII:S0968-0004(97)01170-5
59
REVIEWS
TIBS 23 - FEBRUARY1998 ,i
ii
,
,~
i.R
,
Table I. Birth defects associated with mutations in fibroblast growth factor receptors" i
|l
i
,i
Syndrome
Gene
i,
i
Typical mutations i
i
Refs
i
FGFR1
Pfeiffer
Igil-lglll linker
9
FGFR2
Crouzon
Mainly in third Ig-like domain. Creation or removal of cysteines, leaving an unpaired number of cysteine residues. Highly conserved amino acids As for Crouzon, sometimes with identical mutations As for Crouzon Igll-lglll linker Missense mutations leading to creation of cysteine residues
5
Pfeiffer Jackson-Weiss Apert Beare Stevenson FGFR3
Craniosynostosis Achondroplasia Crouzon with acanthosis nigricans Hypochondroplasia Thanatophoric dysplasia I Thanatophoric dysplasia II Skeletal-Skin-Brain syndrome (SSB)
Igll-lglll linker region Transmembrane Gly380Arg Transmembrane, Ala391Glu Tyrosine kinase domain Asn540Lys Missense mutations leading to creation of cysteine residues Tyrosine kinase domain Lys650Glu Tyrosine kinase domain Lys650Met
26 27 7 28 10, 11 12, 13 14 29 30 30 31
,ll
"Abbreviations: FGFR, fibroblast growth factor receptor; Ig, immunoglobulin.
mutations at equivalent positions have been detected in more than one of the FGFRs (Table U). It is striking that not only is the position of the amino acid conserved, but also the nature of the replacement, suggesting strongly that these particular changes will have quite specific effects in altering the action of the receptors. This is discussed below, together with the probable mode of action, in the context of a proposed structural model of the molecule. Two classes of mutation are particularly revealing. The most highly conserved motif in the Ig super-family is the presence of two cysteine residues stabilizing
the extracellular domains (see Fig. 2). Both cysteines in the third lg domain of FGFR2 are frequently observed to be mutated 5,6with the possibility of the creation of an unbound cysteine, which could result in ligand-independent dimerization of receptor molecules (through formation of an intermolecular disulphide bond), leading to constitutive activation. This prediction is reinforced by the finding of further mutations outside the lg-like domains in FGFR2 and FGFR3, in which a tyrosine or serine is mutated to cysteine. The region between the second and third lg-likedomains is strongly conserved between members of the FGFR family
Box 1. Clinical descriptions of birth defects Involving mutations in FGFR genes Craniosynostosis, which affects approximately i in 2500 children, is the premature fusion of the skull bone sutures. Various syndromic forms have been defined on the basis of observation of craniosynostosis in association with other malformations. Amongst these are the autosomal dominantly inherited syndromes of Apert, Crouzon, Pfeiffer and Jackson-Weiss (all named after the clinician originally describing them). Apert syndrome is characterized by the syndactyly (fusion of digits), both bony and cutaneous, of the hands and feet, while a diagnosis of Crouzon syndrome depends on the very striking degree of proptosis (prominent eyes) in the absence of limb abnormalities, contrasting with broad thumbs and halluces (big toes) seen in Pfeiffer syndrome. Jackson-Weiss syndrome is less easy to characterize and the associated malformations vary between individuals. Acanthosis nigricans is a velvety thickening and darkening of the skin, in this case around the shoulders and elbows. Beare Stevenson cutis gyrata syndrome involves craniosynostosis, acanthosis nigricans plus furrowed skin. It is associated with early death. Achondroplasia is the most frequent form of short-limb dwarfism. Thanatophoric dysplasia (TD) is a more severe form in which the ribs, as well as the arms and legs, are shortened. Cases are classified into subtypes based on the presence of curved as opposed to straight femurs; patients with straight, relatively long femurs always had associated severe cloverleaf skull and were designated TD type II (TD2), while TD cases with curved, short femurs with or without cloverleaf skull were called TD type I (TD1). This clinical distinction has proved valid at the molecular level. Hypochondroplasia is a milder form of achondroplasia. The newly described Skeletal-Skin-Bone (SSB) syndrome is characterized by short stature, profound developmental delay and acanthosis nigricans. Further details of all these disorders may be found in the online version of Mendelian Inheritance in Man (OMIM), http://www.ncbi.nlm.nih.gov/omim/
60
and between different species. It might, therefore, be expected that the amino acids within this region act in a highly specific manner, and the mutations found support this idea. Just two mutations within this region (Ser252Trp and Pro253Arg) account for all but one case of Apert syndrome, to date r. In the other case, Ser252 is mutated to phenylalanine, the amino acid most structurally similar to tryptophan~. The equivalent mutation, Pro252Arg of FGFR1, which arises in some cases of Pfeiffer syndrome'J, and Pro250Arg of F'GFR3, has been found in a wide range of patients with craniosynostosis. The latter includes some with an initial diagnosis of Crouzon, Pfeiffer, isolated craniosynostosis, non-syndromic craniosynostosis or Saethre-Chotzen syndrome ~0Jl. Two specific mutations have been found within the transmembrane region of FGFR3, both involving a change in charge at position 380; the neutral amino acid glycine is changed to the more basic arginine in nearly all cases of achondroplasia ~2Ja and a mutation of alanine to glutamic acid, only 11 amino acids away, results in a form of Crouzon syndrome together with acanthosis nigricans (a thickened and velvety appearance of the skin) ~4,~5. It is revealing to plot the mutations on a predicted model of the second and third Ig-like domains in the extracellular region of FGFR2 (see Fig. 2), which is similar in most respects to the model of Gray et al. 1~ (see Figure legend) and to the alignment of Bateman and Chothia ~;. Not only do mutations involving unpaired cysteines abound, but modelling shows that several of the other reported mutations 4 are predicted to destabilize the disulphide bridge, also leading to the possibility of unpaired cysteine residues. For example, the invariant Trp290 residue in the C-strand of lglll is found at the centre of the region between the two 13-sheets and stabilizes the Cys-Cys bond. The mutation Trp290Arg, which we have found as a recurrent mutation in patients, is likely to lead to the disruption of the packing of the core of the domain and reduce its stability. Another common mutation involves the invariant tyrosine residue (Tyr340His) which lies adjacent to the conserved Cys342 in the hydrophobic core. Disruption of the tertiary structure by either of these mutations could lead to difficulties in forming the intradomain disulphide bridge, possibly resulting in alternative intermolecular bonds. Mutations leading directly to the creation of a free cysteine have been found in other regions of FGFR2 and FGFR3,
TIBS 2 3 -
REVIEWS
FEBRUARY1998
including the linker region between lgl] and ]g]H and in the transmembrane region. In Fig. 2, the mutations within [gill (i.e. Ser347Cys and Ser351Cys) are shown in blue and are predicted to be in an exposed loop between strands F and (;1~.17. Because they are exposed, they are more likely to lead to Cys-Cys crosslinking than disruption of disulphide bridging between the two sheets of the ]g domains. The mutations in the linker region (Arg248Cys, Ser249Cys of FGFR3), which cause thanatophoric dysplasia, probably cause ligand-independent dimedzation of the receptors and provide a marked contrast to the mutation Pro250Arg, found in craniosynostosis, which probably affects FGF binding (see below)Z~. Functional studies
A number of studies have looked at the effect of some of the disease-causing mutations on FGFR function. The overall picture is that the receptors become partially or constitutively activated, with ligand-indepe~ldent dimerization and autophosphorylation ~-':~. The degree of activation of the signalling, as measured by the degree of phosphorylation, appears to correlate well with the clinical severity of the mutation'-"-'. Xenopus iaevis oocytes provided a useful experimental system to study the effect of various mutations. In two separate experiments, mutations were introduced into the related Xenopus FGFR1 and FGFR2 genes (XFGFR1, XFGFR2)1~.19. RNA was transcribed in vitro from mutagenized plasmids and injected into oocytes. The cells were induced to differentiate similarly to the FGFl-mediated induction of wild-type cells. Those mutations that involved the creation of an unpaired cysteine (analogous to Cys278Phe or Cys342Tyr of FGFR2) resulted in the covalent dimerization of receptor molecules, through the formation of intermolecular disulphide bonds and subsequent ]igandindependent enhanced tyrosine-kinase activity ~9. Double mutations (analogous to Cys278Phe and Cys342Tyr of FGFR2) resulted in a monomeric species. A transmembrane mutation (analogous to Gly380Arg of FGFR3) and a tyrosine-kinasedomain mutation (analogous to Lys650Glu of FGFR3) all showed increased tyrosinekinase phosphorylation. However, a mutation within the linker region, Prol60Arg (analogous to the important Pro252Arg found in all three FGFRs) did not increase tyrosine phosphorylation, but did exhibit increased FGF1 and FGF2 binding compared with the wild type. It is likely that the human
Table il. Amino acid mutations found in corresponding positions in mete than one FGFR
Amino acid~
FGFR1
FGFR2
FGFR3
Refs
Pro253Arg Ser372Cys Tyr375Cys Gly384Arg
Pfeiffer
Apert Beare Stevenson Beare Stevenson Craniosynostosis
Craniosynostosis Thanatophoric dysplasia Thanatophoric dysplasia Achondroplasia
9.7.10 28.30 28.30 32
aNumbering is based on the sequence of FGFR2, Abbr~v at nn FGFR fihrnbla~t p.rowth factor receptor.
mutations within the lgll-lglll linker follow this pattern and result in alteration of FGF ligand specificity, rather than tyrosine phosphorylation. It seems that the genetic mutations act in a dominant-negative fashion, either by constitutive activation or by alteration of ligand/receptor specificity. This is in line with the dominant pattern of inheritance and with the observation that no mutations that predict the loss of a copy of the gene product or a truncated protein have been detected in either FGFR1, 2 or 3. Indeed, of all the possible codon changes which can alter Cys, six have
been seen, leading to substitution of either Tyr, Phe, Set, Arg or Trp. Strikingly the mutation TGC to TGA (Cys to stop) has not been found. Children with WolfHirschhorn syndrome, which is a chromosomal disorder arising from the loss of the tip of the short arm of chromosome 4, have no associated growth abnormalities, even though there is complete removal of a copy of FGFR3. Further evidence in support of this theory comes from the creation of FGFR~ deficient mice, constructed by targeted disruption of the FGFR3 gene. These mice have longer-than-average bones '~:'~
'
Thr268
Arg330
Thr'319
Ala362 Pro253 a
N-term
Cys278
Tyr34o
L
Ser351
Gin285
Ser347
Pro303
Figure 2 A ribbon diagram of a comparative model of the third immunoglobulin (Iglll) extracellular domain with the 'c' splice form of FGFR2. This domain, and the equivalent domains within FGFR1, have previously been modelled by analogy to IgV domains 16, IgC domains 33, IgC-like domains 34 and IgV-like domains35. However, the domain has been shown to possess wellconserved features of the recently defined I-set Ig family17. These include the conserved cysteine residues Cys278 and Cys342, the invariant tryptophan residue Trp290, and the conserved tyrosine residue Tyr340. We have constructed the model using the COMPOSER36 suite of programs (Sybyl; Tripes Inc., USA) by analogy with the crystal structure 37 of telokin, the C-terminal domain of myosin lighL-chain kinase from turkey gizzard. Telokin is one of only three members of the I-set Ig family of proteins whose structure has been characterized. Telokin was chosen because it gave the best amino acid sequence alignment with FGFR2, with the fewest insertions or deletions. This is in agreement with Bateman and Chothia 1T, who analysed the sequence similarities of each of the three extracetlular Ig demains of FGFR2 and concluded that each of the domains contained all the major secondary structure elements found in telokin. The domain consists of two four-stranded beta-sheets (ABED and A'GFC). The conserved residues, which are often mutated and cause skeletal disorders, are shown. The picture was produced using the program SETOR38.
61
TIBS 2 3 - F E B R U A R Y 1 9 9 8
REVIEWS which suggests that FGFR3 is a negative regulator of bone growth. Because the mutations causing short stature (achondroplasia and thanatophoric dysplasia) increase the ligand-independent activity of FGFR3, it fits well that these mutations act by overcoming the negative control normally exerted by FGFR3.
Conclusion Initially it might seem surprising that mutations that lead to the failure of correct formation of skull sutures or of bone growth are, in fact, the result of the ligand-independent activation of receptor kinases. However, this does fit with the predominance of mutations that result in either the creation of unpaired cysteines or destabilization of a disulphide bond. Some transmembrane mutations probably form stable interactions, such as hydrogen bonds, between transmembrahe helices of two receptors, thus leading to dimerization. F,xceptions to this are mutations in the linker region between lgll and Iglll that probably result in an alteration of ligand-binding properties. Genetic studies have revealed that the FGFRs function to balance cell growth versus differentiation in the skull and limbs, with activation leading to differentiation and skeletogenesis. Different members of the FGFR family are synthesized differently in time and space. Mutations in FGFR1, 2 or 3 can overlap in the clinical picture in the patient, so that they can apparently compensate for each other to a surprising extent, and seem to have overlapping functions. The studies to date have highlighted the crucial importance of certain amino acids within the IglI-lglli linker region, probably because they control the specificity of FGF binding. Because mutations have not yet been identified for approyJmately half the patients with Crouzon and Pfeiffer syndrome, other critical regions remain to be discovered.
34(8), 632-636 12 Shiang. R. et al. (1994) Cell 78, 335-342 13 Rousseau, E et al. (1994) Nature 371,
252-254 14 Meyers, G. A. et al. (1995) Nat. Genet. 111, 111 15 Wilkes, D. et al. (1996) J. Med. Genet. 33, 744-748 16 Gray, l. E. et al. (1995) Biochemistry 34, 10325-10333 17 Bateman, A. and Chothia, C. (1995) Nat.
EAS'/TOfl~'/T~AT
Struct. Biol. 2,
1068-1074
L'Ehb WON~ II~Kki I N~ ASL.,b,
18 Neilson, K. M. and
Friesel, R. E (1995) J. Biol. Chetn. 270,
26037-26040 /9 Neilson, K. M. and Friesel, R. E. (1996) J. Biol. Chem. 271, 25049-25057 20 Galvin, B. D. et aL (1996) Poc. Natl. Acad. Sci. U. S. A. 93, 7894-7899 21 Webster, M. K. and Donoghue, D. J. (1996) EMBO J. 15, 520-527 22 Naski, M. C. et aL (1996) Nat. Genet. 13, 233-237 23 Li, Y. et al. (1997) Oncogene 14, 1397-1406 24 Colvin. J. S. et aL (1996) Nat. Genet. 12, 390-397 25 Deng, C. et al. (1996) Cell 84, 911-921 26 Rutland, P. et al. (1995) Nat. Genet. 9, 173-176 27 Jabs, E. W. et al. (1994) Nat. Genet. 8, 275-284 28 Przylepa, K. A. et al. (1996) Nat. Genet. 13. 492-494 29 Bellus, G. A. et aL (1995) Nat. Genet 10, 357-359 30 Tavormina, P. L. et al. (1995) Nat. Genet. 9, 321-330 31 Francomano, C, A. et aL (1996) Am. J. Hum. Genet. 59, A25 32 Pulleyn, L. J. et al. (1996) Eur. J, Hum. Genet. 4, 283-291 References 33 Wang, F. et al. (1995) i Johnson, D. E. and Williams, L. T. (1993) Adv. J. Biol. Chem. 270, Cancer Res. 60, 1-41 10222-10230 2 Dionne,C. A. et aL (1990) EMBO1. 9, 2685-2692 3 Sternberg M, J. and Gullick, W. J. (1990) Protein 34 Pantoliano. M. W. et al. (1994) Biochemistry Eng. 3, 245-248 33. 10229-10248 4 Webster, M. K. and Donoghue, D. J. (1997) 35 Xu, J. et al. (1992) Trends Genet. 13, 178-182 J. Biol. Chem. 267, 5 Reardon,W. et aL (1994) Nat. Genet. 8, 98-103 17792-17803 60tdridge, M. et al. (1995) Hum. Mol. Genet. 4, 36 Srinivasan, N. and 1077-1082 Blundell, T. L, (1993) 7 Wilkie, A. O. M. et al. (1995) Nat. Genet. 9, Protein Eng. 6, 501 165-172 37 Holden, H. M. et al. 80ldridge, M. et al. (1997) Hum. 114o1.Genet. 6, (1992) J. Mol. Biol. 137-143 227, 840--851 9 Muenke. M. et al. (1994) Nat. Genet. 8, 269-273 38 Evans, S. V. (1993) 10 Be,us, G. A. et al. (1996) Nat. Genet. 14, J. MoL Graph, 11, 174-176 134-138 11 Reardon, W. et al. (1997) J. Med. Genet.
62
ii
-Tii~= AL¢I~Lfl~I~5]~ N~NE-~'L~NSE I¢ ~ E
b¢~ IAEVAL
AL(HEI~y
STI~PF
0~
:if!i: ~ "
.--I~ ¢~.-FEcT0F'A/
~NA~ l~nfl Wm~E~ I~N~ l,v't~A~S~ j tOffEf~ED J-rtI¢
Pete Jeffs is a freelancer working in Paris, France.
TIBS 2 3 - FEBRUARY1998
Fbrob]ast growth factor recepbrs: le,,sons from the genes Dav)d Burke, David Wilkes, Tom L. Blundetl and Sue Malcolm The fibroblast growth factor receptors (FGFRs) are a family of transmembrahe tyrosine kinases involved in signalling via interactions with the family of fibroblast growth factors (FGFs). Genetic findings have provided a way of dissecting these interactions. Mutations in three members of the FGFR family have been found in patients with birth defects involving craniosynostosis (premature fusion of the cranial sutures) or skeletal abnormalites. Analyses of the spectrum of mutations found predict that many of them will result in ligand-independent activation of the receptors. Amino acids have also been identified that are likely to be important in determining the specificity of FGFR-FGF interactions. VARIOUS METHODS have proved popular in attempts to translate biochemical findings about gene products into an understanding of the function of these genes in different tissues or the whole organism. Traditionally, mice with the gene knocked-out have been created and Drosophila melanogastermutants identified. In these studies, the gene in question is often rendered inactive, but in human developmental disorders the complete removal of the gene product arising from recessive or null mutations are the exception, rather than the rule. Gene function is often altered by a dosage effect, involving the loss of one copy (haploinsufficiency), or altered activity (gainof-function) as described here. Naturally occurring mutations provide an exceptional opportunity to study the different consequences of altered genes.
and survival, and have been implicated in pathological processes including anglogenesis, wound healing and cancer. The FGFRs interact with fibroblast growth factors (FGFs), of which at least 13 members have been characterized to date. Consequently, the interactions are extremely complex and difficult to disentangle by biochemical techniques ~. The FGF ligands interact with the extracellular portion of FGFR molecules, which consists of three immunoglobulin (lg)-like domains with short linking segments (Fig. 1). In the segment between Igl and lgll there is a run of the acidic residues g!utamic acid and aspartic acid,
(a)
D. Burke and T. Blundell are at the Department of Biochemistry, Universityof Cambridge, Tennis Court Road, Cambridge,UK CB2 1QW. D. Wilkes and S. Malcolm are at the Molecular Genetics Unit, Institute of Child Health, 30 Guilford Street, London, UK WC1N 1EH. Email: [email protected]
Igll
r~utations associated with birth defects A number of birth defects are associated with mutations in FGFRs Gable 1) which often involve incorrect fusion of the skull sutures or skeletal abnormalities. Box i describes the characteristics of the syndromes listed in Table I. Although uumerous different mutations haw been observed (over 25 in Crouzon syndrome alone), there is a distinct clustering in terms of type of mutation and position within the FGFR molecules (.see Ref. 4). One of the most striking features is the extent to which homologous
Iglll TM
ND The FGFRfamily The FGFRfamily consists of four closely related members, whose amino acid sequences are highly conserved between different members of the family and throughout evolution. They regulate a multitude of cellular processes, including cell growth, differentiation, migration
Igl
which is referred to as the 'acid box', as well as a three-amino acid motif hisfidinealanine-valine (HAV). The HAV sequence is also found in the cell-adhesion molecules N-CAM, L-CAM and N-cadherin, and it is thought to be involved in binding between molecules. There is a single transmembrane portion and, inside the cell, there is a split tyrosine-kinase domain. The overall complexity is increased by the existence of additional forms of the receptor, created by alternative splicing of mRNA. These receptors are either missing the first Ig domain (lgl), missing both Igl and the acid-box region or, more importantly, possess an alternative sequence for the C-terminal half of the third ]g-like domain encoded by a separate exon ~ (shaded black in Fig. la). Interaction between growth factor, receptor and heparan sulphate proteoglycans is required for activation of signalling. After ligand binding, receptor molecules dimerize and this is followed by autophosphorylation of several tyrosine residues in the intracelluar domain 3.
C-s-s-C--~
sP
Acid box
C-s-s-C
TK1-TK2
C-,.s-s-C
/\
j Pro253Arg
Ser372Cys Tyr375Cys
Gly384Arg
(b) FGFR2-111b
314 H S G I N S S - - N A E V L A L F ~ r T E A D A G E Y I C K V S N Y I G Q A N Q S A W L T V L P
359
FGFR2-111c
314 A A ~ ; V N T T D K E I E V L Y I R N V T F E D A G E Y T C L A G N S I G I S F H S A W L T V L P
361
Figure 1
(a) The fibroblast growth factor receptor, consisting of: the N-terminal signal peptide (SP); the acid-box region (a run of acidic residues); the three imrnunoglobulin-likedomains (Igl, Igll and Iglll); the transmernbrane region (TM); and the split bjrosine-kinase domain (TK1-TK2). The two cysteines forming the disulphide bridge are shown in each Ig-like domain. The dark region in Iglll is explained in the text. (b) Sequencealignmentof the alternative forms of the C-terminalhalf of the Igll[ domain (Iglllb and Iglllc) of FGFR2.The sequences shown represent the complete exon. Identical residues are shown in purple.
Copyright © 1998,ElsevierScience Ltd. All rights reserved. 0968-0004/98/$19.00 PII:S0968-0004(97)01170-5
59
REVIEWS
TIBS 23 - FEBRUARY1998 ,i
ii
,
,~
i.R
,
Table I. Birth defects associated with mutations in fibroblast growth factor receptors" i
|l
i
,i
Syndrome
Gene
i,
i
Typical mutations i
i
Refs
i
FGFR1
Pfeiffer
Igil-lglll linker
9
FGFR2
Crouzon
Mainly in third Ig-like domain. Creation or removal of cysteines, leaving an unpaired number of cysteine residues. Highly conserved amino acids As for Crouzon, sometimes with identical mutations As for Crouzon Igll-lglll linker Missense mutations leading to creation of cysteine residues
5
Pfeiffer Jackson-Weiss Apert Beare Stevenson FGFR3
Craniosynostosis Achondroplasia Crouzon with acanthosis nigricans Hypochondroplasia Thanatophoric dysplasia I Thanatophoric dysplasia II Skeletal-Skin-Brain syndrome (SSB)
Igll-lglll linker region Transmembrane Gly380Arg Transmembrane, Ala391Glu Tyrosine kinase domain Asn540Lys Missense mutations leading to creation of cysteine residues Tyrosine kinase domain Lys650Glu Tyrosine kinase domain Lys650Met
26 27 7 28 10, 11 12, 13 14 29 30 30 31
,ll
"Abbreviations: FGFR, fibroblast growth factor receptor; Ig, immunoglobulin.
mutations at equivalent positions have been detected in more than one of the FGFRs (Table U). It is striking that not only is the position of the amino acid conserved, but also the nature of the replacement, suggesting strongly that these particular changes will have quite specific effects in altering the action of the receptors. This is discussed below, together with the probable mode of action, in the context of a proposed structural model of the molecule. Two classes of mutation are particularly revealing. The most highly conserved motif in the Ig super-family is the presence of two cysteine residues stabilizing
the extracellular domains (see Fig. 2). Both cysteines in the third lg domain of FGFR2 are frequently observed to be mutated 5,6with the possibility of the creation of an unbound cysteine, which could result in ligand-independent dimerization of receptor molecules (through formation of an intermolecular disulphide bond), leading to constitutive activation. This prediction is reinforced by the finding of further mutations outside the lg-like domains in FGFR2 and FGFR3, in which a tyrosine or serine is mutated to cysteine. The region between the second and third lg-likedomains is strongly conserved between members of the FGFR family
Box 1. Clinical descriptions of birth defects Involving mutations in FGFR genes Craniosynostosis, which affects approximately i in 2500 children, is the premature fusion of the skull bone sutures. Various syndromic forms have been defined on the basis of observation of craniosynostosis in association with other malformations. Amongst these are the autosomal dominantly inherited syndromes of Apert, Crouzon, Pfeiffer and Jackson-Weiss (all named after the clinician originally describing them). Apert syndrome is characterized by the syndactyly (fusion of digits), both bony and cutaneous, of the hands and feet, while a diagnosis of Crouzon syndrome depends on the very striking degree of proptosis (prominent eyes) in the absence of limb abnormalities, contrasting with broad thumbs and halluces (big toes) seen in Pfeiffer syndrome. Jackson-Weiss syndrome is less easy to characterize and the associated malformations vary between individuals. Acanthosis nigricans is a velvety thickening and darkening of the skin, in this case around the shoulders and elbows. Beare Stevenson cutis gyrata syndrome involves craniosynostosis, acanthosis nigricans plus furrowed skin. It is associated with early death. Achondroplasia is the most frequent form of short-limb dwarfism. Thanatophoric dysplasia (TD) is a more severe form in which the ribs, as well as the arms and legs, are shortened. Cases are classified into subtypes based on the presence of curved as opposed to straight femurs; patients with straight, relatively long femurs always had associated severe cloverleaf skull and were designated TD type II (TD2), while TD cases with curved, short femurs with or without cloverleaf skull were called TD type I (TD1). This clinical distinction has proved valid at the molecular level. Hypochondroplasia is a milder form of achondroplasia. The newly described Skeletal-Skin-Bone (SSB) syndrome is characterized by short stature, profound developmental delay and acanthosis nigricans. Further details of all these disorders may be found in the online version of Mendelian Inheritance in Man (OMIM), http://www.ncbi.nlm.nih.gov/omim/
60
and between different species. It might, therefore, be expected that the amino acids within this region act in a highly specific manner, and the mutations found support this idea. Just two mutations within this region (Ser252Trp and Pro253Arg) account for all but one case of Apert syndrome, to date r. In the other case, Ser252 is mutated to phenylalanine, the amino acid most structurally similar to tryptophan~. The equivalent mutation, Pro252Arg of FGFR1, which arises in some cases of Pfeiffer syndrome'J, and Pro250Arg of F'GFR3, has been found in a wide range of patients with craniosynostosis. The latter includes some with an initial diagnosis of Crouzon, Pfeiffer, isolated craniosynostosis, non-syndromic craniosynostosis or Saethre-Chotzen syndrome ~0Jl. Two specific mutations have been found within the transmembrane region of FGFR3, both involving a change in charge at position 380; the neutral amino acid glycine is changed to the more basic arginine in nearly all cases of achondroplasia ~2Ja and a mutation of alanine to glutamic acid, only 11 amino acids away, results in a form of Crouzon syndrome together with acanthosis nigricans (a thickened and velvety appearance of the skin) ~4,~5. It is revealing to plot the mutations on a predicted model of the second and third Ig-like domains in the extracellular region of FGFR2 (see Fig. 2), which is similar in most respects to the model of Gray et al. 1~ (see Figure legend) and to the alignment of Bateman and Chothia ~;. Not only do mutations involving unpaired cysteines abound, but modelling shows that several of the other reported mutations 4 are predicted to destabilize the disulphide bridge, also leading to the possibility of unpaired cysteine residues. For example, the invariant Trp290 residue in the C-strand of lglll is found at the centre of the region between the two 13-sheets and stabilizes the Cys-Cys bond. The mutation Trp290Arg, which we have found as a recurrent mutation in patients, is likely to lead to the disruption of the packing of the core of the domain and reduce its stability. Another common mutation involves the invariant tyrosine residue (Tyr340His) which lies adjacent to the conserved Cys342 in the hydrophobic core. Disruption of the tertiary structure by either of these mutations could lead to difficulties in forming the intradomain disulphide bridge, possibly resulting in alternative intermolecular bonds. Mutations leading directly to the creation of a free cysteine have been found in other regions of FGFR2 and FGFR3,
TIBS 2 3 -
REVIEWS
FEBRUARY1998
including the linker region between lgl] and ]g]H and in the transmembrane region. In Fig. 2, the mutations within [gill (i.e. Ser347Cys and Ser351Cys) are shown in blue and are predicted to be in an exposed loop between strands F and (;1~.17. Because they are exposed, they are more likely to lead to Cys-Cys crosslinking than disruption of disulphide bridging between the two sheets of the ]g domains. The mutations in the linker region (Arg248Cys, Ser249Cys of FGFR3), which cause thanatophoric dysplasia, probably cause ligand-independent dimedzation of the receptors and provide a marked contrast to the mutation Pro250Arg, found in craniosynostosis, which probably affects FGF binding (see below)Z~. Functional studies
A number of studies have looked at the effect of some of the disease-causing mutations on FGFR function. The overall picture is that the receptors become partially or constitutively activated, with ligand-indepe~ldent dimerization and autophosphorylation ~-':~. The degree of activation of the signalling, as measured by the degree of phosphorylation, appears to correlate well with the clinical severity of the mutation'-"-'. Xenopus iaevis oocytes provided a useful experimental system to study the effect of various mutations. In two separate experiments, mutations were introduced into the related Xenopus FGFR1 and FGFR2 genes (XFGFR1, XFGFR2)1~.19. RNA was transcribed in vitro from mutagenized plasmids and injected into oocytes. The cells were induced to differentiate similarly to the FGFl-mediated induction of wild-type cells. Those mutations that involved the creation of an unpaired cysteine (analogous to Cys278Phe or Cys342Tyr of FGFR2) resulted in the covalent dimerization of receptor molecules, through the formation of intermolecular disulphide bonds and subsequent ]igandindependent enhanced tyrosine-kinase activity ~9. Double mutations (analogous to Cys278Phe and Cys342Tyr of FGFR2) resulted in a monomeric species. A transmembrane mutation (analogous to Gly380Arg of FGFR3) and a tyrosine-kinasedomain mutation (analogous to Lys650Glu of FGFR3) all showed increased tyrosinekinase phosphorylation. However, a mutation within the linker region, Prol60Arg (analogous to the important Pro252Arg found in all three FGFRs) did not increase tyrosine phosphorylation, but did exhibit increased FGF1 and FGF2 binding compared with the wild type. It is likely that the human
Table il. Amino acid mutations found in corresponding positions in mete than one FGFR
Amino acid~
FGFR1
FGFR2
FGFR3
Refs
Pro253Arg Ser372Cys Tyr375Cys Gly384Arg
Pfeiffer
Apert Beare Stevenson Beare Stevenson Craniosynostosis
Craniosynostosis Thanatophoric dysplasia Thanatophoric dysplasia Achondroplasia
9.7.10 28.30 28.30 32
aNumbering is based on the sequence of FGFR2, Abbr~v at nn FGFR fihrnbla~t p.rowth factor receptor.
mutations within the lgll-lglll linker follow this pattern and result in alteration of FGF ligand specificity, rather than tyrosine phosphorylation. It seems that the genetic mutations act in a dominant-negative fashion, either by constitutive activation or by alteration of ligand/receptor specificity. This is in line with the dominant pattern of inheritance and with the observation that no mutations that predict the loss of a copy of the gene product or a truncated protein have been detected in either FGFR1, 2 or 3. Indeed, of all the possible codon changes which can alter Cys, six have
been seen, leading to substitution of either Tyr, Phe, Set, Arg or Trp. Strikingly the mutation TGC to TGA (Cys to stop) has not been found. Children with WolfHirschhorn syndrome, which is a chromosomal disorder arising from the loss of the tip of the short arm of chromosome 4, have no associated growth abnormalities, even though there is complete removal of a copy of FGFR3. Further evidence in support of this theory comes from the creation of FGFR~ deficient mice, constructed by targeted disruption of the FGFR3 gene. These mice have longer-than-average bones '~:'~
'
Thr268
Arg330
Thr'319
Ala362 Pro253 a
N-term
Cys278
Tyr34o
L
Ser351
Gin285
Ser347
Pro303
Figure 2 A ribbon diagram of a comparative model of the third immunoglobulin (Iglll) extracellular domain with the 'c' splice form of FGFR2. This domain, and the equivalent domains within FGFR1, have previously been modelled by analogy to IgV domains 16, IgC domains 33, IgC-like domains 34 and IgV-like domains35. However, the domain has been shown to possess wellconserved features of the recently defined I-set Ig family17. These include the conserved cysteine residues Cys278 and Cys342, the invariant tryptophan residue Trp290, and the conserved tyrosine residue Tyr340. We have constructed the model using the COMPOSER36 suite of programs (Sybyl; Tripes Inc., USA) by analogy with the crystal structure 37 of telokin, the C-terminal domain of myosin lighL-chain kinase from turkey gizzard. Telokin is one of only three members of the I-set Ig family of proteins whose structure has been characterized. Telokin was chosen because it gave the best amino acid sequence alignment with FGFR2, with the fewest insertions or deletions. This is in agreement with Bateman and Chothia 1T, who analysed the sequence similarities of each of the three extracetlular Ig demains of FGFR2 and concluded that each of the domains contained all the major secondary structure elements found in telokin. The domain consists of two four-stranded beta-sheets (ABED and A'GFC). The conserved residues, which are often mutated and cause skeletal disorders, are shown. The picture was produced using the program SETOR38.
61
TIBS 2 3 - F E B R U A R Y 1 9 9 8
REVIEWS which suggests that FGFR3 is a negative regulator of bone growth. Because the mutations causing short stature (achondroplasia and thanatophoric dysplasia) increase the ligand-independent activity of FGFR3, it fits well that these mutations act by overcoming the negative control normally exerted by FGFR3.
Conclusion Initially it might seem surprising that mutations that lead to the failure of correct formation of skull sutures or of bone growth are, in fact, the result of the ligand-independent activation of receptor kinases. However, this does fit with the predominance of mutations that result in either the creation of unpaired cysteines or destabilization of a disulphide bond. Some transmembrane mutations probably form stable interactions, such as hydrogen bonds, between transmembrahe helices of two receptors, thus leading to dimerization. F,xceptions to this are mutations in the linker region between lgll and Iglll that probably result in an alteration of ligand-binding properties. Genetic studies have revealed that the FGFRs function to balance cell growth versus differentiation in the skull and limbs, with activation leading to differentiation and skeletogenesis. Different members of the FGFR family are synthesized differently in time and space. Mutations in FGFR1, 2 or 3 can overlap in the clinical picture in the patient, so that they can apparently compensate for each other to a surprising extent, and seem to have overlapping functions. The studies to date have highlighted the crucial importance of certain amino acids within the IglI-lglli linker region, probably because they control the specificity of FGF binding. Because mutations have not yet been identified for approyJmately half the patients with Crouzon and Pfeiffer syndrome, other critical regions remain to be discovered.
34(8), 632-636 12 Shiang. R. et al. (1994) Cell 78, 335-342 13 Rousseau, E et al. (1994) Nature 371,
252-254 14 Meyers, G. A. et al. (1995) Nat. Genet. 111, 111 15 Wilkes, D. et al. (1996) J. Med. Genet. 33, 744-748 16 Gray, l. E. et al. (1995) Biochemistry 34, 10325-10333 17 Bateman, A. and Chothia, C. (1995) Nat.
EAS'/TOfl~'/T~AT
Struct. Biol. 2,
1068-1074
L'Ehb WON~ II~Kki I N~ ASL.,b,
18 Neilson, K. M. and
Friesel, R. E (1995) J. Biol. Chetn. 270,
26037-26040 /9 Neilson, K. M. and Friesel, R. E. (1996) J. Biol. Chem. 271, 25049-25057 20 Galvin, B. D. et aL (1996) Poc. Natl. Acad. Sci. U. S. A. 93, 7894-7899 21 Webster, M. K. and Donoghue, D. J. (1996) EMBO J. 15, 520-527 22 Naski, M. C. et aL (1996) Nat. Genet. 13, 233-237 23 Li, Y. et al. (1997) Oncogene 14, 1397-1406 24 Colvin. J. S. et aL (1996) Nat. Genet. 12, 390-397 25 Deng, C. et al. (1996) Cell 84, 911-921 26 Rutland, P. et al. (1995) Nat. Genet. 9, 173-176 27 Jabs, E. W. et al. (1994) Nat. Genet. 8, 275-284 28 Przylepa, K. A. et al. (1996) Nat. Genet. 13. 492-494 29 Bellus, G. A. et aL (1995) Nat. Genet 10, 357-359 30 Tavormina, P. L. et al. (1995) Nat. Genet. 9, 321-330 31 Francomano, C, A. et aL (1996) Am. J. Hum. Genet. 59, A25 32 Pulleyn, L. J. et al. (1996) Eur. J, Hum. Genet. 4, 283-291 References 33 Wang, F. et al. (1995) i Johnson, D. E. and Williams, L. T. (1993) Adv. J. Biol. Chem. 270, Cancer Res. 60, 1-41 10222-10230 2 Dionne,C. A. et aL (1990) EMBO1. 9, 2685-2692 3 Sternberg M, J. and Gullick, W. J. (1990) Protein 34 Pantoliano. M. W. et al. (1994) Biochemistry Eng. 3, 245-248 33. 10229-10248 4 Webster, M. K. and Donoghue, D. J. (1997) 35 Xu, J. et al. (1992) Trends Genet. 13, 178-182 J. Biol. Chem. 267, 5 Reardon,W. et aL (1994) Nat. Genet. 8, 98-103 17792-17803 60tdridge, M. et al. (1995) Hum. Mol. Genet. 4, 36 Srinivasan, N. and 1077-1082 Blundell, T. L, (1993) 7 Wilkie, A. O. M. et al. (1995) Nat. Genet. 9, Protein Eng. 6, 501 165-172 37 Holden, H. M. et al. 80ldridge, M. et al. (1997) Hum. 114o1.Genet. 6, (1992) J. Mol. Biol. 137-143 227, 840--851 9 Muenke. M. et al. (1994) Nat. Genet. 8, 269-273 38 Evans, S. V. (1993) 10 Be,us, G. A. et al. (1996) Nat. Genet. 14, J. MoL Graph, 11, 174-176 134-138 11 Reardon, W. et al. (1997) J. Med. Genet.
62
ii
-Tii~= AL¢I~Lfl~I~5]~ N~NE-~'L~NSE I¢ ~ E
b¢~ IAEVAL
AL(HEI~y
STI~PF
0~
:if!i: ~ "
.--I~ ¢~.-FEcT0F'A/
~NA~ l~nfl Wm~E~ I~N~ l,v't~A~S~ j tOffEf~ED J-rtI¢
Pete Jeffs is a freelancer working in Paris, France.