Structural and Functional Diversity in the FGf Receptor Multigene Family

STRUCTURAL AND FUNCTIONAL DIVERSITY IN THE FGF RECEPTOR MULTIGENE FAMILY Daniel E. Johnson and Lewis T. Williams Howard Hughes Medical Institute, Program of Excellence in Molecular Biology, and Cardiovascular Research Institute, University of California, San Francisco, California 94143-0724

I. Introduction

11. The FGF Family of Polypeptide Mitogens

111. IV. V. VI. VII.

VIII.

IX.

X. XI. XII. XIII. XIV. XV. XVI. XVII. XVIII. XIX.

Early Binding and Cross-Linking Studies of the FGF Receptor Nomenclature of the FGF Receptor Genes Purification, cDNA Cloning, and Characterization of FGFR 1 Characterization of FGFR 2, FGFR 3, and FGFR 4 Multiple Forms of FGFR 1 and FGFR 2 Are Generated by Alternative Splicing Multiple Forms of FGFR 1 A. Variations Involving Ig Domain I B. The Inclusion or Exclusion of Two Amino Acids in the Extracellular Domain C. Three Alternative Exons for the Second Half of Ig Domain 111 Multiple Forms of FGFR 2 A. Variations Involving Ig Domain I B. Variations Involving the Acid Box Domain C. Alternative Exons for the Second Half of Ig Domain 111 D. The Inclusion or Exclusion of Two Amino Acids in the Juxtamembrane Domain E. Three Alternative Exons for the C-Tail Domain FGFR 3 and FGFR 4 Ligand Binding Specificities of the Cloned FGF Receptors Alternative Splicing in the Third Ig Domain Is Important for Determining Ligand Binding Specificities Analogous Splice Variants from Different FGF Receptor Genes Encode Receptor Forms with Different Ligand Binding Specificities Regulation of FGF Receptor Expression Cell- and Tissue-Specific Alternative Splicing of FGF Receptor mRNAs A. Cell- and Tissue-Specific Alternative Splicing of the Third Ig Domain B. Tissue-Specific Alternative Splicing Involving Ig Domain I Differential, Tissue-Specific Expression of the Different FGF Receptor Genes The Drosophila FGF Receptor FGF Receptor-Mediated Signal Transduction Concluding Remarks References

1 ADVANCES IN CANCER RESEARCH, VOL. 60

Copyright 8 1993 by Academic Press. Inc. All rights of reproduction in any form reserved.

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DANIEL E. JOHNSON AND LEWIS T. WILLIAMS

1. introduction

The fibroblast growth factors (FGFs) constitute a family of closely related polypeptide mitogens (Burgess and Maciag, 1989; Folkman and Klagsbrun, 1987; Gospodarowicz et al., 1986b; Thomas, 1987). Currently, seven members of this family have been identified on the basis of amino acid sequence homologies. T h e FGF family has distinguished itself from other growth factor families by virtue of the pleiotropic actions of its members. In addition to their abilities to stimulate proliferation of a wide variety of cells, FGFs exhibit potent neurotrophic and angiogenic activities (R.S. Morrison et al., 1986; Walicke et al., 1986; Folkman and Klagsbrun, 1987; Anderson et al., 1988). FGFs also have the capacity to induce differentiation, inhibit differentiation, or maintain a differentiated phenotype of cells in culture (Linkhart et al., 1981; Serrero and Khoo, 1982; Broad and Ham, 1983; Lathrop et al., 1985; Togari et al., 1985; Wagner and DAmore, 1986; Anderson et al., 1988). Furthermore, a substantial body of evidence indicates that FGFs play important roles during development. The effects of FGFs are known to be mediated by high affinity receptor tyrosine kinases. T h e recent purification and cDNA cloning of FGF receptors have led to the discovery of a family of structurally related FGF receptor molecules. Within this family there is an enormous degree of complexity. Four distinct FGF receptor genes have been identified, and in the case of at least two of these genes, multiple mRNA transcripts are known to be generated by alternative splicing. A number of studies have now shown that the structurally diverse receptor molecules are also functionally different. Moreover, characterization of structural and functional diversity within the FGF receptor family is beginning to shed new light on differences in the mechanisms of action among members of the FGF familv.

II. The FGF Family of Polypeptide Mitogens The first members of the FGF family to be purified and characterized were acidic FGF (aFGF) and basic FGF (bFGF). Both factors were purified on the basis of their mitogenicity toward fibroblasts, using bovine pituitary (Armelin, 1973; Gospodarowicz et al., 1974; Gospodarowicz, 1975) and brain (Trowel1 et al., 1939; Hoffman, 1940; Gospodarowicz et al., 1978) as sources. In subsequent studies aFGF and bFGF were purified from a wide variety of sources including adrenal gland (Gospodarowicz et al., 1986a), bone (Hauschka et al., 1986), cartilage (Sullivan

FGF RECEPTOR MULTIGENE FAMILY

3

and Klagsbrun, 1985), corpus luteum (Gospodarowiczet al., 1985a), hypothalamus (Klagsbrun and Shing, 1985), kidney (Baird et al., 1985a), liver (Ueno et al., 1986), placenta (Gospodarowiczet al., 1985b), prostate (Nishi et al., 1985), retina (Baird et al., 1985b), testis (Ueno et al., 1987), and thymus (Gospodarowiczet al., 1986b).A significant improvement in the purification of aFGF and bFGF was made when it was discovered that both factors bind to heparin (Maciag et al., 1984; Shing et al., 1984). This led to the development of standardized purification protocols using heparin affinity chromatography (Shing et al., 1983; Lobb et al., 1986). The highly purified preparations of FGFs were subjected to amino acid sequencing, and this ultimately made possible the cloning of cDNAs for both aFGF (Gimenez-Gallegoet al., 1985; Thomas et al., 1985;Jaye et al., 1986) and bFGF (Esch et al., 1985; Abraham et al., 1986a,b; Kurokawa et al., 1987). The cloned cDNAs for aFGF and bFGF each encode proteins of 155 amino acids. Curiously, the predicted amino acid sequences of both factors do not contain signal peptide sequences. In this respect, aFGF and bFGF resemble the interleukin-1 (IL1) proteins, which also lack signal peptide sequences. Despite considerable attention to this issue, it remains unclear how growth factors without signal peptides are secreted from the cell. The predicted sequences for human aFGF and human bFGF are 55% identical at the amino acid level. Both proteins are also highly conserved across species (Thomas, 1987). Human and bovine aFGF differ by only 12 amino acids, and human and bovine bFGF differ by only 2 amino acids. The proteins also share limited homology to IL1-a and IL1-p (25-27% amino acid identity). The cDNAs encoding aFGF and bFGF are derived from distinct, single-copy genes. The human gene encoding aFGF is located on chromosome 5 Uaye et al., 1986),whereas the human bFGF gene is located on chromosome 4 (Mergia et al., 1986).Despite their different chromosomal loci, the FGF genes have a similar structural organization. Both genes are composed of three exons, and contain two large introns at similar locations (Mergia et al., 1986; Abraham et al., 198613). Biological studies using purified or recombinant aFGF and bFGF have shown that both factors are potent mitogens for a wide variety of cells of mesenchymal and neuroectodermal origin (Burgess and Maciag, 1989; Gospodarowicz et al., 1986b). Of particular interest has been the discovery that both factors act as mitogens and chemoattractants for endothelial cells in uitro (Burgess and Maciag, 1989), and exhibit potent angiogenic activity in uzuo (Folkman and Klagsbrun, 1987).Hence, FGFs may play an important role in the normal development of the vascular

4

DANIEL E. JOHNSON A N D LEWIS T. WILLIAMS

system through their action on endothelial cells. At the same time, however, aberrant production of FGFs or other aberrations in FGF response pathways may contribute to pathological conditions that result from either too much or too little vascularization. Thus, it is important to consider the potential involvement of FGFs in the vascularization of tumors, in wound healing, and in vascular diseases such as diabetic retinopathy. FGFs also stimulate cellular production of collagenases and plasminogen activator (Gross et al., 1982, 1983; Presta et al., 1985; Moscatelli et al., 1986; Mignatti et al., 1989), and this could help potentiate neovascularization or tumor invasiveness (Folkman and Klagsbrun, 1987). FGFs may also lead to cellular transformation through autocrine or paracrine mechanisms. Several groups have demonstrated that constitutive expression of exogenous FGF in transfected cells promotes growth in serum-free media and soft agar, and is tumorgenic in mice. The results that are obtained in these experiments, however, vary with the host cell line that is used. With some cell lines, certain parameters of transformation are highly dependent on the secretion of FGF Uaye et al., 1988; Rogelj et al., 1988; Sasada et al., 1988),whereas with other cell lines transformation is independent of FGF secretion (Neufeld et al., 1988; Jaye et al., 1988). In addition to their ability to promote cellular proliferation, FGFs also influence the differentiation of a variety of cell types. FGFs induce the differentiation of preadipocyte fibroblasts into adipocytes (Serrero and Khoo, 1982; Broad and Ham, 1983) and stimulate neurite outgrowth from hippocampal neurons (Walicke et at., 1986), cerebral cortical neurons (R. S. Morrison et al., 1986), and rat PC12 cells (Togari et al., 1985; Wagner and D’Amore, 1986). While both factors can act to induce a differentiated phenotype, they can also act to inhibit differentiation. This is the case in skeletal muscle myoblasts, where addition of FGFs acts to inhibit differentiation to myotubes (Linkhart et al., 1981; Lathrop et al., 1985).FGFs also support the survival of lesioned cholinergic neurons in vivo (Anderson et al., 1988), indicating a role in the maintenance of differentiated cells. The ability of FGFs to influence the differentiation of a variety of cell types suggests that these factors may play important roles during development. This idea is supported by experiments showing that addition of‘ FGFs to Xen@ embryos leads to mesoderm induction (Kimelman and Kirschner, 1987; Slack et al., 1987). The presence of FGF in early Xen0fni.s embryos indicates that FGFs may serve in this capacity during normal embryogenesis (Kimelman et al., 1988). Indeed, recent experiments have shown that selective disruption of FGF receptor-mediated

FGF RECEPTOR MULTIGENE FAMILY

5

signaling pathways in developing Xenopus embryos leads to dramatic inhibition of mesoderm formation and developmental defects in gastrulation and posterior development (Amaya et al., 1991). Over the last several years, five additional members of the FGF family have been identified on the basis of amino acid sequence homologies, These proteins are approximately 35 to 45% identical with aFGF and bFGF and include the product of the int-2 oncogene (Moore et al., 1986), the product of the hst oncogene (Kaposi sarcoma FGF) (Taira et al., 1987; Bovi et al., 1987), FGF-5 (Zhan et al., 1988), FGF-6 (Marks et al., 1989), and keratinocyte growth factor (KGF; Finch et al., 1989; Rubin et al., 1989). In addition to sequence homologies, these proteins also share some physical and biological properties with aFGF and bFGF, such as the ability to bind heparin and the ability to stimulate proliferation of a variety of cells of mesenchymal and neuroectodermal origin (Burgess and Maciag, 1989). In contrast to aFGF and bFGF, however, int-2 hstlKFGF, FGF-5, FGF-6, and KGF contain signal peptide sequences encoded by their mRNA transcripts. The int-2 oncogene was originally identified as a preferred site of integration for mouse mammary tumor virus (Peters et al., 1983). Mice containing viral integration near the int-2 gene frequently display transcriptional activation of the int-2 oncogene and develop mammary carcinomas (Peters et al., 1983, Dickson et al., 1984; Moore et al., 1986).The hst oncogene was identified in a focus forming assay following transfection of 3T3 cells with genomic DNA from a human stomach cancer cell line (Taira et al., 1987). The same gene was identified when DNA from Kaposi sarcoma cells was used to transfect cells (Bovi et al., 1987).We will refer to this gene as the hstlKFGF gene. FGF-5 was identified as a human gene whose fortuitous rearrangement during DNA transfection led to focus formation in 3T3 cells (Zhan et al., 1988). cDNAs for FGF-6 were isolated on the basis of nucleic acid hybridization with a hstlKFGF probe (Marks et al., 1989). Keratinocyte growth factor was cloned using amino acid sequence data obtained from the purified protein (Finch et al., 1989; Rubin et al., 1989). The FGFs are known to exert their effects by binding to high affinity receptors on the surface of responsive cell types. The existence of multiple members of the FGF family has raised the question whether all members of this family bind to a common receptor molecule. Alternatively, there could be multiple FGF receptors, each possessing different ligand binding properties. Recent advances in the FGF receptor field have shown that both of these possibilities are partially correct.

6

DANIEL E. JOHNSON AND LEWIS T. WILLIAMS

Ill. Early Binding and Cross-Linking Studies of the FGF Receptor Early characterization of FGF receptors focused on binding and cross-linking studies using iodinated aFGF or bFGF. These studies revealed that a variety of cells express saturable high affinity receptors with Kds of 50-500 pM and 10-200 pM for aFGF (Schreiber et al., 1985; Baird et al., 1986; Libermann el al., 1987) and bFGF (Neufeld and Gospodarowicz, 1985; Moenner et al., 1986; Olwin and Hauschka, 1986; Moscatelli, 1987), respectively. Competition analyses demonstrated that either ligand was able to compete for high affinity binding of the other ligand (Neufeld and Gospodarowicz, 1986; Olwin and Hauschka, 1986). This provided the first indication that aFGF and bFGF might share a common receptor. A related study has also shown that hstlKFGF causes downregulation of high affinity binding sites for bFGF, indicating that hstIKFGF also binds to the same receptor (Moscatelli and Quarto, 1989). When either aFGF or bFGF was used in cross-linking experiments, receptor species in the range 125-165 kDa were detected on SDS-polyacrylamide gels (G. Neufeld and Gospodarowicz, 1985; G. Neufeld and Gospodarowicz, 1986; Friesel et al., 1986; Moenner et al., 1986; Olwin and Hauschka, 1986, 1989; Libermann et al., 1987; Courty et al., 1988). Frequently two prominent cross-linked bands of 125 and 145 kDa were seen. Initially it was thought that the 125-kDa protein represented a proteolytic cleavage product of the 145-kDa protein. Recent evidence, however, indicates that the two proteins are derived from different alternatively spliced forms of the FGF receptor mRNA (see discussion in Section VII1,A). As expected, either ligand was able to block the crosslinking of the same or different radiolabeled ligand to both the 125-kDa and the 145-kDa proteins (Neufeld and Gospodarowicz, 1986; Olwin and Hauschka, 1986). In summary, the results of binding and crosslinking studies strongly supported the hypothesis that different members of the FGF family shared a common receptor. The idea of a common receptor for all members of the FGF family did not fit, however, with data obtained for KGF. This growth factor, although potently mitogenic for epithelial cells, failed to stimulate the growth of endothelial cells and fibroblasts, cells that are responsive to both aFGF and bFGF (Rubin et al., 1989). Thus, KGF did not appear capable of binding to the receptors for aFGF and bFGF that are present on these cells (Bottaro et al., 1990). As will be described in further sections, the KGF receptor represents a unique splice variant of an FGF receptor gene. Before the discussion on the binding of FGFs to cell surface receptors

7

FGF RECEPTOR MULTIGENE FAMILY

is concluded, it should be noted that cells also express a large number of low affinity binding sites for FGFs (Neufeld and Gospodarowicz, 1985; Moenner et al., 1986; Olwin and Hauschka, 1986; Clegg et al., 1987; Moscatelli, 1987). Considerable evidence indicates that the low affinity sites represent heparan sulfate proteoglycan molecules located on the cell surface or in the extracellular matrix (Moscatelli, 1987, 1988; Bashkin et al., 1989). Binding to low affinity sites occurs with a Kd of 2 to 10 nM and can be removed by incubation with heparin, washing with 2.0 M NaC1, or treatment with heparinase (Moscatelli, 1987). Recent evidence indicates that the binding of FGFs to low affinity sites plays a role in potentiating binding of FGFs to high affinity receptors (Rapraeger et al., 1991; Yayon et al., 1991). This information has recently been reviewed elsewhere (Klagsbrun and Baird, 1991)and will not be discussed further. Instead, this review will focus on the high affinity cell surface FGF receptors. IV. Nomenclature of the FGF Receptor Genes

Since the isolation of the first complete FGF receptor cDNA in 1989 (Lee et al., 1989),our understanding of the complexity of the FGF receptor field has increased dramatically. To date, four distinct FGF receptor genes have been identified. Furthermore, in the case of at least two of these genes it is clear that alternative splicing gives rise to multiple forms of the receptor. The different FGF receptor genes and splice variants of these genes are described in the literature using many different names (see Table I and Fig. 5). In an effort to simplify our understanding of TABLE I NOMENCLATURE OF THE DIFFERENT FGF RECEPTORGENES FGFR 1

FGFR 2

FGFR 3

FGFR 4

"g bFGFR Cekl N-bFGFR h2, h3, h4, h5 FGFR 1

bek Cek3 K-Sam K-Sam K-Sam' TK14 TK25 KGFR FGFR 2

Cek2 FGFR 3

FGFR 4

Note. The table shows some of the names that have been used to describe the different FGF receptor genes and cDNAs derived from alternatively spliced FGF receptor mRNA transcripts. The publications that were used to compile this list are referenced in the text.

8

DANIEL E. JOHNSON AND LEWIS T. WILLIAMS

FGF receptor diversity, we will refer to the different FGFR genes as FGFR 1, FGFR 2, FGFR 3, and FGFR 4, in the chronological order in which they were first identified and characterized. V. Purification, cDNA Cloning, and Characterization of FGFR 1

The first FGF receptor (FGFR 1) to be characterized in detail was purified from chicken embryos. In the purification scheme that was used, extracts from chicken embryos were incubated with biotinylated FGF in the presence of heparin (Lee et al., 1989). Biotin-FGF/FGFR complexes were then purified on an avidin-agarose column. Different ligand-affinity purification schemes have been employed by other laboratories (Imamura et al., 1988; Burrus and Olwin, 1989). The inclusion of heparin in the original purification protocol appears to have been critically important for purifying the high affinity receptor, as other approaches that have not included heparin have resulted in the isolation of low affinity binding proteins (Kiefer et al., 1990). The amino acid sequence obtained from peptides of the purified chicken FGF receptor showed striking similarity with the predicted amino acid sequences of two previously published partial cDNA clones: humanflg (Ruta et al., 1988)and mouse bek (Kornbluth et al., 1988).The human flg (fm-like gene) cDNA had been isolated from an endothelial cell cDNA by virtue of it hybridization with a c-fm probe. The region of cross-hybridization of this probe is presumably in the coding sequence for the tyrosine kinase domain of flg; otherwise JEg and c$m are not homologous. The mouse bek (bacterially expressed kinase) cDNA had been isolated from a cDNA expression library by probing with antiphosphotyrosine antibodies. Although the function of the flg and bek proteins was unknown at the time of the discovery of their partial cDNAs, it has since come to light that the full-length fig and bek cDNA clones represent specific splice variants of the FGFR 1 and FGFR 2 genes, respectively (see Sections V I I I and IX, and Fig. 5). A cDNA encoding the chicken FGFR 1 was isolated using oligonucleotide probes based on the amino acid sequence from the purified protein (Lee et al., 1989). The cDNA encoded a protein with a deduced molecular mass of 92 kDa (not including carbohydrate side chains) that contained several features commonly found in growth factor receptors (see Fig. 1). The protein contained a single membranespanning region, an amino-terminal signal peptide, and three extracellular immunoglobulin-like (Ig-like) domains (Williams and Barclay, 1988). Between the first (I) and the second (11) Ig-like domain, the

FGF RECEPTOR MULTIGENE FAMILY

9

FIG. 1. Schematic diagram of the chicken FGFR 1 structure. The following structural features are identified: hydrophobic leader sequence (striped box), three extracellular Iglike domains (labeled I, 11, and 111), acid box domain (open box), transmembrane domain (solid box), kinase 1 and kinase 2 domains (stippled boxes).

receptor contained a unique domain that has not been seen in other growth factor receptors. This domain consists of eight consecutive acidic residues and is referred to as the “acid box.” The intracellular domain of the FGFR 1 protein contained consensus tyrosine kinase sequences. This confirmed earlier biochemical evidence which indicated that the receptor was a tyrosine-specific protein kinase (Huang and Huang, 1986; Coughlin et al., 1988).The tyrosine kinase sequence of FGFR 1 is split by an insertion of 14 amino acids. The length of this kinase insert region is considerably shorter than those of the PDGF-P (Yarden et al., 1986) and CSF-1 (Coussens et al., 1986) receptors (104 and 70 amino acids, respectively), but comparable to those of the insulin and insulin-like growth factor-1 receptors (Ullrich et al., 1985, 1986). Another interesting feature of the chicken FGFR 1 protein is the length of the juxtamembrane domain. This domain consists of the region between the transmembrane domain and the first kinase domain (kinase 1). The FGFR 1juxtamembrane domain is 79 amino acids long, compared with juxtamembrane domains of 49 to 5 1 amino acids for the PDGF-f3 (Yarden et al., 1986),CSF-1 (Coussens et al., 1986),EGF (Ullrich et al., 1984), HER1 (Coussens et al., 1985), and insulin (Ullrich et al., 1985) receptors. Complementary DNA clones encoding similar FGFR 1 forms have subsequently been isolated from a variety of species including human (Dionne et a,!., 1990; Johnson et al., 1990; Eisemann et al., 1991; Hou et al., 1991), mouse (Mansukhani et al., 1990; Reid et al., 1990; Werner et al., 1992a),chicken (Pasquale and Singer, 1989),and Xenopus (Musci et al., 1990). The degree of amino acid identity between FGFR 1 proteins from different species is striking (see Fig. 2). Overall, when compared to the human FGFR 1 protein, the mouse, chicken, and Xenopls FGFR 1 proteins are 98, 91, and 78% identical, respectively. The most highly conserved regions of the receptor molecule are the kinase 1 and kinase 2 domains (92 and 95% identity, respectively, between human and Xenopus). The least conserved regions are the signal peptide region (50%, human to Xenopw), Ig domain I (54%), the membrane-proximal

10

DANIEL E. JOHNSON AND LEWIS T. WILLIAMS

HUMAN

vs

MOUSE

HUMAN

vs

HUMAN

vs

CHICKEN XENOPUS

Signal peptide

93 %

70

Yo

50 Yo

lg domain I

93 Yo

80 Yo

54 Yo

97

Oh

83 %

73 Yo

100 Yo

95 %

79 Yo

97 Yo

98 Yo

79 Yo

Membraneproximal TM

100 % 90 %

75 Yo 86 %

44 Yo

JM

100 Yo

92 Yo

81 Yo

Kinase 1

100 Yo

99 Yo

92 Yo

93

Oh

71 Yo

50 Yo

99 %

99 Yo

95 Yo

95 Yo

80 %

75 Yo

98 %

91 Yo

78 Yo

(119)

Acid box

(149)

Ig domain I1 (248)

Ig domain 111

Kinase insert Kinase 2

C-tail

57 Oh

FIG.2. Comparison of FGFR 1 proteins from different species. The figure shows the degree of amino acid identity between different domains of the human FGFR 1 protein (Dionne et al., 1990) and the corresponding domains of FGFR 1 proteins from mouse (Reid ~t al., 1990), chicken (Lee et al., 1989). and Xenopvs (Musci et al., 1990). All of the proteins compared represent 3 Ig domain receptor forms containing IIIc-type sequences in the third Ig domain. The numbers in parentheses indicate the last amino acid of each domain

FGF RECEPTOR MULTIGENE FAMILY

11

domain (44%), the transmembrane domain (57%),and the kinase insert domain (50%).

VI. Characterization of FGFR 2, FGFR 3, and FGFR 4 Following the isolation of the chicken FGFR 1 cDNA, cDNAs derived from three additional FGFR genes (2,3,and 4) were isolated and characterized by several laboratories. The proteins encoded by these genes are structurally similar to the FGFR 1 protein and are highly conserved at the amino acid level. Comparison of the mouse FGFR 1 sequence and the partial sequence of mouse bek revealed several differences at the amino acid and nucleotide levels. The isolation of full-length bek clones led to the discovery that bek was in fact the product of a different gene (Dionne et al., 1990). We will refer to this gene as FGFR 2 for the remainder of this review. Additional FGFR 2 cDNAs have been isolated as cDNAs hybridizing with amplified DNA fragments from a human stomach cancer cell line (Hattori et al., 1990). Also, cDNA clones encoding the receptor for keratinocyte growth factor, a unique splice variant of FGFR 2 (see further sections), were isolated using a novel expression cloning strategy (Miki et al., 1991). Even more FGFR 2 cDNA clones have been isolated using PCR or library screening (Saiki et al., 1988; Pasquale, 1990; Houssaint et al., 1990; Champion-Arnaud et al., 1991; Crumley et al., 1991; Raz et al., 1991; Sat0 et al., 1991; Dell and Williams, 1992). Complementary DNA clones derived from a third FGFR gene (FGFR 3) were obtained by low stringency screening of a human K-562 (chronic myelogenous leukemia cell line) cDNA library with a v-sea oncogene probe (Keegan et al., 1991). Human cDNA clones derived from a fourth

in the human FGFR 1 protein (numbering based on Fig. 3). The signal peptide region contains signal peptide sequences as well as sequences between the signal peptide and the beginning of Ig domain I. The boundaries of Ig domain I, the acid box domain, and Ig domains I1 and 111 were defined by intron/exon junctions (see Fig. 6 and Johnson et al., 1991). The boundaries of the membrane-proximal domain were defined by the introdexon junction at the end of Ig domain 111 (N-terminus) and the beginning of the transmembrane domain (C-terminus). The boundaries of the juxtamembrane domain were defined by the end of the transmembrane domain (N-terminus) and the beginning of the kinase 1 domain (C-terminus). The boundaries of the kinase 1, kinase insert, and kinase 2 domains were defined by comparison with consensus kinase sequences as reported by Hanks et al. (1988). Sequences following the end of the kinase 2 domain constitute the C-tail domain.

FGFRl MWSWKCLLFWAVLVTAT--LCTARPSPTLPEQAQ------PWGAPVEVESFLVH-PGDLL FGFRZ .V..GRFICLV.VTM..--.SL....FS.V.DTTLEPEEP.TKYQISQPEVY.AA..ES. FGFR3 .GRPA.A.ALC.A.AIVAGASSESLGTEQRWGRAAEVPG.EPGQQ.Q---..FGS..AV FGFR4 .RLLLA..GVLLS.PGPPV.SLEASEEVEL.PCLA-----.SLEQQEQELT-VA-L.QPV

51 58 57 53

FGFRl QLRCRLRDDV--QSINWLRDGVQLAESNRTRITGEEVEVQDSVPADSGLYACVTSSPSGS FGFR2 EV . . L.K .AA--- V.S.TK ...H.GPN ...VLI ..YLQ1KGAT.R....... TA.RTVD. FGFR3 E.S.PPPGGGPMGP TV.VX.. TG .VP. E.VLVGPQRLP.LNASHE...A.S.RQRLTQRV FGFR4 R.-.CG.AERG---GH.YKE.SR..PAG.V.GWRGRL.IASFL.E.A.R.L.LARGSMIV

109 115 117 109

FGFRl FGFR2 FGFR3 FGFR4

DTTYFSVNVSDALPSSEDDDDDDDSSSEE-KETDNTKPNRMPVAPYWTSPEKMEKKLHAV E.W ..M ...T..I-..G..E..T.GAE.FVS.NS.NS.N.R-----.....NT.....R.... LC-H ...R.T ..-...G..E.GE.EAEDTGVD.G--------- .....R..R.D ...L.. LQ-NLTL1TG.S.T ..N ..E.PK-.HRDPSNRHSYPQQ----- .....H.QR

........

168 169 166 162

FGFRl PAAKTVKFKCPSSGTPNPTLRWLKNGKEFKPDHRIGGYKVRYATWSIIMDSWPSDKGNY 228 F G F R ~ ...N ....R..AG.N.M..M..........QE.........NQH..L..E.......... 229 FGFR3 . . .N ..R . R . . A A . N.T.SIS .....R..RGE.....I.L.HQQ..LV.E......R... 226 FGFR4 . .GN ....R . . A A . N.T ..I....D.QA.HGEN....IRL.HQH..LV.E......R. T. 222 FGFRl FGFRZ FGFR3 FGFR4

.....

288 289 206 282

FGFRl WLKHIEVNGSKIGPDNLPWQILKTAGVNTTDKEMEVLHLRSFEDAGEYTCLAGNSIG FGFRZ .I..V.K .... Y...G...LKV..A. ........ I...YI...T................ FGFR3 .... V . . . . . . V...GT...TV.....A ...... L...S.H..T................ FGFR4 .....VI ... SF.AVGF....V. ...DI.SS--.V...Y.....A.... ...........

348 349 346 340

FGFRl FGFR2 FGFR3 FGFR4

LSHHSAWLTVLEALEER-PAVMTSPLYLEIIIYCTGAFLISCMVGSVIVYKMKSGTKKSD

1.F ........ P.PGRE-KEITA..D ....A...I.V...A...VT..LCR..NT...P. F . . . . . . . V..P.E..LVE.DEAGSV.AG.LS.GV.F..FILV.AA.TLCRLR.PP.. GL . .YQ .......PEEDPTWT.AAPEAR.TD..L.AS.SLALAVLLLLAGL.RGQALHGRHP

407 408 406 400

FGFRl FGFW FGFR3 FGFR4

FHSQMAVHKLAKSIPLRRQIPPVSADSSASMNSGVLLVR-PSRLSS-SGTPMLAGVSEYEL .S . .P.....T.R...........E..S....NTP...ITT....TAD............ --GSPT IS-RF.. K...SLESNA.--.S.NTP...I-A....-GEG.T..N...L.. -RPPAT.Q..S-RF..A..FSLESG..G--K.SSS...-GV....-..PAL...LVSLD.

465 468 459 454

FGFRl FGFR2 FGFR3 FGFR4

PEDPRWELPRDRLVLGKPLGEGCFGOVVLAEAIGLDKDKPL

525 528 519 514

TCIVENEYGSINHTYQLDVVERSPHRPILQAGLPANKTVALGSNVEFMCKVYSDPQPHIQ . .V H....................ASTW.GD...V......A..... V ...KF RQ T. ..L................Q.AV...D...H......A..... L...AV...RYN.L. ..L...... T . A W D..LL...... A

............ .. ... .. ..

..........

..

...

....K ..F...K.T..............M...V.I.....KEAVT.......D.......

.A..K...S.A..T..............M.....I...RAAKPVT.......D...D... .L..L..F....................R...F.M.PAR.DQAST.......DN.SD...

FIG.3. The amino acid sequences of four human FGF receptor proteins derived from distinct receptor genes. The amino acid sequences of the human FCFR 2 protein (Dionne ~t al., 1990), the human FGFR 3 protein (Keegan el al., 1991), and the human FGFR 4 protein (Partanen et al., 1991) are shown in comparison to the sequence of the human FGFR 1 protein (Dionne el al., 1990). Each sequence represents a 3 Ig domain receptor form containing IIIc t y p e sequences in the third Ig domain. For FGFRs 2, 3, and 4, only sequences which differ from the FGFR 1 sequence are shown. Dashed lines indicate gaps that have been introduced into the sequence.

13

FGF RECEPTOR MULTIGENE FAMILY

FGFRl SDLISEMEMMKMIGKHKNIINLLGACTQDGPLWIVEYASKGNLREYLQAPGLEYCY FGFR2 V... R ......M.. S. FGFR3 ...V G.....L....A......F.R....... D.SF FGFR4 A..V....V ..L..R .........V ...E........C.A......F.R...... PDLSP

...

......................................... ........................

585 588 579 574

FGFRl FGFR2 FGFR3 FGFR4

NPSHNPEEQLSSKDLVSCAYQVARGUEYLASKKCIHRDLAAFUWLVTEDNVMKIADFGLA 645 648 DINRV ....MTF ......T ..L.........Q................N........... DTCKP TF.. Q. 639 DGPRSS.GP ..FPV Q E.R................ ............ 634

FGFRl FGFR2 FGFR3 FGFR4

RDIHHIDYYKKTTNGRLPVKWMAPEALFDRIYTHQSDVWSFGVLLWEIFTLGGSPYPGVP V M............. I. V.NL V................. ..........I. .GV S.................V...........I............... I.

.....

................. ........................... ............ ..

............................. .. ........................ .........

.............

705 708 699 694

FGFRl VEELFKLLKEGHRMDKPSNCTNELYMMMRDCWHAVPSQ~TFKQLVEDLDRIVALTSNQE 765 LT..T. E. 768 FGFR2 .................A HD. ..I..E. ...A................VLTV..T D. 759 FGFR3 .................A E. 753 FGFR4 .....S ..R......R.PH.PP...GL..E....A............A..KVL-.AVS

.................................. ...

FGFRl FGFR2 FGFR3 FGFR4

YLDLSMPLDQYSPSFPDTRSSTCSSGEDSVFSHEPLPEEPCLPRHPAQLANGGLKRR .....Q ..E.....Y....-.S....D.....PD.M.Y.....QY.HI--.. S-VKT .....A.FE ....GGQ..P-.SS...D....AHDL..----- .AP.SS---. .S-.T ....RLTFGP ....GG.AS-.....S- ......D...--LGSSSF.F----.SGVQT

822 821 806 802

FIG.3. (cont.)

FGFR gene (FGFR 4)were isolated using PCR with tyrosine kinase specific primers followed by library screening with the amplified fragments (Partanen et al., 1991). The amino acid sequences of 3 Ig domain forms of human FGFR 2, FGFR 3, and FGFR 4 proteins in comparison to the human FGFR 1 protein are shown in Fig. 3. Table I1 shows the overall level of amino TABLE I1 COMPARISON OF THE DIFFERENT HUMANFGF RECEPTORGENES FGFR 1 FGFR 2 FGFR 3

FGFR 2 72%

FGFR 3 62% 66%

FGFR 4 55% 57% 61%

Note. The table shows the overall degree of amino acid identity between the four amino acid sequences shown in Fig. 3. All of the proteins compared represent 3 Ig domain receptor forms containing lIIc type sequences in the third Ig domain. Percentage identity was calculated by dividing the number of identities by the number of amino acids in the larger of the two receptor proteins being compared.

14

DANIEL E. JOHNSON AND LEWIS T. WILLIAMS

FGFR 1

FGFR 1

FGFR 1

FGFR 2

FGFR 3

FGFR 4

43 Yo

17 Yo

20 %

40

Yo

27 Yo

19 Yo

43

Yo

33 Yo

27

79 Yo

64 %

61 Yo

78 Oh

81 %

74

Yo

38 62

31 Yo 33 Yo

13 24

Yo Yo

76 Yo

46

39 Yo

88%

83 Yo

75 Yo

50 Yo

43 Yo

7 Yo

62 Yo

46 %

42 Yo

vs

Signal peptide

q-

(30)

Ig domain I

vs

vs

(119)

Acid box Ig domain I1

(149)

-.=

Yo

(248)

Ig domain I11 Membrane-

lPiloxima' JM

Kinase 1 Kinase insert

C-tail

I

(360) (376) (397)

. _..: ...... ........ ...... .__._. ......... ;:.:. ..... ........ :.:...... ...... ........ .:::._

(476)

...... ..... .._._. ..... ..... _..: ......

(580)

.... ........

(594)

...... .:....... ....... ..... .,I._ .: ... ......

FIG.4. Comparison of human FGF receptor proteins derived from four different receptor genes. The figure shows the degree of amino acid identity between different domains of the human FGFR 1 protein (Dionne et nl., 1990) and the corresponding domains of human FGFR 2 (Dionne et al., 1990). human FGFR 3 (Keegan et al., 1991), and humall FGFR 4 (Partanen et al., 1991). All of the proteins compared represent 3 Ig domain receptor forms containing 1IIc-type sequences in the third Ig domain. The numbers in parentheses indicate the last amino acid of each domain in the human FGFR 1 protein (numbering based on Fig. 3). Domain boundaries were defined as described in the legend tor Fig. 2.

FCF RECEPTOR MULTIGENE FAMILY

15

acid identity between the different human FGFR gene products, and Fig. 4 shows a domain by domain comparison of all four human receptor proteins. Overall, the proteins encoded by the four different human genes are strikingly similar. The most closely related proteins are FGFR 1 and FGFR 2 (72% amino acid identity), whereas FGFR 1 and FGFR 4 are the least closely related (55% identity). It is interesting to note that these levels of identity are considerably higher than those observed among different members of the FGF family (35 to 55%). A comparison of the domains of the different human FGF receptors (Fig. 4) reveals a pattern of conservation similar to that observed when comparing FGFR 1 domains across species (Fig. 2). The most highly conserved regions of the different human proteins are the kinase 1 and kinase 2 domains (75 and 84% identity, respectively, between FGFR 1 and FGFR 4). The least conserved regions are the signal peptide region (20%), Ig domain I (19%), the membrane-proximal domain (13%), the transmembrane domain (24%),and the kinase insert domain (7%).

VII. Multiple Forms of FGFR 1 and FGFR 2 Are Generated by Alternative Splicing A striking discovery in the FGF receptor field has been the isolation by several groups of multiple, distinct cDNAs encoding variant forms of FGFR 1 and FGFR 2 (Dionne et al., 1990;Johnson et al., 1990; Reid et al., 1990; Champion-Arnaud et al., 1991; Eisemann et al., 1991; Hou et al., 1991; Miki et al., 1991). Evidence obtained from studies of the organization of the FGFR 1 and FGFR 2 genes indicates that alternative splicing of mRNA is responsible for generating the diverse receptor forms (Champion-Arnaud et al., 1991; Johnson et al., 1991). Although alternative splicing is not unprecedented in the growth factor receptor field, the sheer number of alternative FGF receptor forms far outweighs that seen for any other growth factor receptor. Figure 5 shows a schematic diagram of the different FGF receptor proteins encoded by FGF receptor cDNAs that have been isolated to date. As shown in the figure, alternative splicing results in either (a) the inclusionlexclusion of additional amino acids or (b) the use of alternate coding exons with no net gain or loss of amino acids. In either case, the resultant proteins are structurally different. In subsequent sections of this review, the different splice variants and the potential function of these variants are discussed. VIII. Multiple Forms of FGFR 1 This section discusses the various regions of diversity that have been observed for FGFR 1, beginning at the amino terminus and proceeding

FGFR 1 1

I

h4

*

FGFR 2 1

iQbFGFR

I K-sarn’

KGFR

K-sarn

~

BEWTKl4

FGFR 3 BEK

TK25

I

FGFR 4

nn

(Vlll)

(xtii)

FIG. 5. Schematic diagram of FGF receptor protein structures. The figure shows the structure of variant receptor forms predicted by published cDNAs. T h e names of some receptor variants as they appear in the literature are written directly above the structure. Although cDNAs encoding the receptor variant depicted by an asterisk (iii) have not been isolated, PCR and Northern blotting experiments have identified mRNA transcripts encoding this receptor form (Johnsonet al., 199 1 ; Werner et al., 1992a). Furthermore, both 3 Ig and 2 Ig domain forms of this receptor mRNA appear to exist. T h e following structural features are identified in the figure: the 32 unique amino acids at the C-terminus of the FGFR 1 Ig domain I secreted form (solid oval), acid box domains (open boxes), alternative sequences for the second half of Ig domain 111 labeled IIIa, IIIb, or IIIc (thick black line), transmembrane domains (solid boxes), kinase 1 and kinase 2 domains (stippled boxes), and the unique C-tail domains of 2 FGFR 2 proteins (checkered box and striped box). The following sources were used to compile this figure: (i, Eisemann et al., 1991),(ii,Johnson et al., 1990), (iv, Lee et al., 1989; Pasquale and Singer, 1989), (v, Johnson e l al., 1990; Mansukhani et al., 1990; Reid el al., 1990). (vi, Champion-Arnaud et al., 1991). (vii, Miki el al., 1991), (viii, Hattori et al., 1990), (ix, Dionne et al., 1990; Houssaint et al., 1990), (x, Champion-Arnaud et al., 1991), (xi, Champion-Arnaud et al., 1991), (xii, Keegan et al., 1991), and (xiii, Partanen et al., 1991). It is predicted that more structures will be added to this figure in the coming years.

FGF RECEPTOR MULTIGENE FAMILY

17

toward the carboxyl terminus. Also, reference will be made to published information regarding the organization of exons and introns in the human FGFR 1 gene (Johnson et al., 1991). A. VARIATIONS INVOLVING IG DOMAIN I The first region of FGFR 1 receptor diversity that was observed was in the first Ig-like domain (I). Although the first reported FGFR 1 cDNA encoded a protein with three Ig domains (Fig. 5, iv), several laboratories have subsequently reported the isolation of cDNAs encoding FGFR 1 proteins that are missing Ig domain I (Fig. 5, ii and v) (Johnson et al., 1990; Mansukhani et al., 1990; Reid et al., 1990).Analysis of the FGFR 1 gene revealed the presence of an intron separating Ig domain I from the remainder of the FGFR 1 coding sequence (Fig. 6) (Johnson et al., 1991). Thus it appears likely that the presence or absence of Ig domain I is mediated by alternative splicing. Binding studies have shown that the 3 Ig domain form of FGFR 1 has affinities of 20-80 pM and 50- 150 pM for aFGF and bFGF, respectively (Dionne et al., 1990; Johnson et al., 1990). Similarly, the 2 Ig domain form of FGFR 1 has affinities of 50 and 100 pM for aFGF and bFGF, respectively (Johnson et al., 1990). Thus, Ig domain I does not appear to be necessary for high affinity binding of aFGF and bFGF. Currently, the function of this domain remains unknown. The existence of FGF receptor forms containing 3 or 2 Ig-like domains helps to explain earlier results obtained from cross-linking studies. As discussed previously (Section 111), two prominent bands of 145

111.

FIG.6. The human FGFR 1 gene. The figure shows the arrangement of introns and exons in the human FGFR 1 gene as described by Johnson et al. (1991). The figure also contains one additional exon that was not included in the original description of the gene (solid oval). Arrows indicate the positions of introns and numbers above the arrows indicate the size of the intron in kilobases. The following structural features are identified: the exon encoding the 32 unique amino acids at the C-tail of the Ig domain I secreted form (solid oval), the acid box domain (open box), the three alternative exons for the second half of Ig domain I11 (thick black line; labeled IIIa, IIIb, or IIIc), stop codons at the end of the secreted and membrane-spanning forms (asterisks), the 3’ nontranslated region that is unique to secreted form mRNA transcripts (dashed line), the transmembrane domain (solid box), and the kinase 1 and kinase 2 domains (stippled boxes).

18

DANIEL E. JOHNSON AND LEWIS

T. WILLIAMS

and 125 kDa were frequently seen when aFGF or bFGF were crosslinked to cell surface receptors. More recent studies have utilized cell lines transfected with cDNAs encoding either 3 Ig or 2 Ig domain forms of the receptor (Dionne el al., 1990; Johnson et al., 1990; Mansukhani et al., 1990). The results of these experiments show that cross-linked receptors containing 3 Ig domains are about 145 kDa in size, while crosslinked receptors containing 2 Ig domains are about 125 kDa in size. Thus it appears likely that the 145- and 125-kDa bands originally detected in cross-linking experiments represent the 3 Ig and 2 Ig receptor forms, respectively. In support of this, coexpression of 3 Ig and 2 Ig mRNA transcripts has been detected in a variety of cell lines using PCR (Johnson et al., 1990; Eisemann et al., 1991), Northern blotting (Reid et al., 1990; Eisemann et al., 1991), and RNase protection analyses (Werner et a!., 1992b).

Another receptor variant involving the first Ig domain has been described by Eisemann et al. (1991). The cDNA isolated by this group encodes a complete signal peptide and a complete Ig domain I, followed by 32 unique amino acids and a stop codon (Fig. 5, i). The 32 unique amino acids are encoded by an separate exon located between the exon for Ig domain I and the exon encoding the acid box domain (Fig. 6). Presumably the protein encoded by this cDNA represents a secreted form of Ig domain I. Although the function of this protein is unknown, it is interesting that similar forms of N-CAM molecules are generated via alternative splicing (Cunningham et al., 1987).

B. THEINCLUSION OR EXCLUSION OF Two AMINO ACIDS IN THE EXTRACELLULAR DOMAIN Several groups have reported the isolation of cDNA clones encoding proteins containing (or missing) two additional amino acids just downstream from the acid box Uohnson et al., 1990; Eisemann et al., 1991). In the human FGFR 1 sequence (Fig. 3), these amino acids correspond to Arg (148) and Met (149). Clones encoding proteins containing these amino acids and clones encoding proteins missing these amino acids have been identified for both the 3 Ig and the 2 Ig receptor forms (Johnson et al., 1990; Eisemann et al., 1991). Analysis of genomic DNA indicates that this dipeptide variation does not reflect an allelic difference. Instead, these amino acids are encoded at the immediate 3' end of the acid box domain exon. Therefore, it seems likely that the inclusion or exclusion of these amino acids results from the use of slightly different splice donor sites at this exon/intron boundary (Eisemann et

FGF RECEPTOR MULTIGENE FAMILY

19

al., 1991).The functional significance of this subtle variation in receptor structure is not known. C. THREE ALTERNATIVE EXONS FOR 111 HALFOF Ig DOMAIN

THE

SECOND

Johnson et al. (1990) have reported the isolation of a human cDNA encoding a protein that is identical to the 2 Ig membrane-spanning form of FGFR 1 until a point approximately halfway through Ig domain 111 (Johnson et al., 1990). At this point, the novel protein (see Fig. 5 , ii) diverges completely and then terminates 79 amino acids downstream. The novel protein does not contain a hydrophobic membrane-spanning domain and represents an additional secreted form of the receptor protein. Further studies have shown that a 3 Ig domain form of this secreted protein also exists. Although the function of the secreted FGF receptor is currently unknown, Duan et al. (1992) have shown that this protein binds bFGF. It is possible that the secreted FGF receptor acts as an extracellular reservoir of FGF, regulating the availability of FGFs to cell surface receptors. In the human FGFR 1 gene, sequences encoding the second half of Ig domain I11 which are unique for the secreted receptor form (labeled exon IIIa in Fig. 6) follow immediately downstream from sequences encoding the first half of Ig domain 111 (Johnson et al., 1991). In contrast, sequences encoding the second half of Ig domain I11 which are associated with the membrane-spanning receptor form (labeled exon IIIc in Fig. 6) are located several kilobases downstream. Also, in the process of sequencing this region of the human gene an additional exon was discovered that is highly homologous to the IIIc exon (labeled exon IIIb in Fig. 6). At the amino acid level, the IIIb and IIIc sequences exhibit 45% amino acid identity. PCR and Northern blotting analyses indicate that this new exon is part of mRNA transcripts encoding a membrane-spanning receptor. Thus, there are three alternative exons for the second half of Ig domain 111. One of these exons (IIIa) is a part of mRNA transcripts encoding a secreted receptor form, while the other two (IIIb and IIIc) are a part of transcripts encoding membrane-spanning receptor forms. These three exons have been named in the order of their linear appearance in the FGFR 1 gene (Fig. 6; Johnson et al,, 1991). As will be discussed shortly, corresponding IIIb and IIIc exons are also seen in the FGFR 2 gene. Furthermore, the IIIb and IIIc sequences confer distinct ligand binding specificities to the membrane-spanning forms of both FGFR 1 and FGFR 2.

20

DANIEL E. JOHNSON AND LEWIS T. WILLIAMS

IX. Multiple Forms of FGFR 2 A. VARIATIONSINVOLVING Ig DOMAIN As is the case for FGFR 1, cDNAs encoding 3 Ig domain and 2 Ig domain forms of FGFR 2 have also been identified (see Fig. 5 ) (Dionne et al., 1990; Hattori et al., 1990; Houssaint et al., 1990; Champion-Arnaud et al., 1991; Crumley et al., 1991; Miki et al., 1991). Binding studies have shown that both forms of FGFR 2 exhibit similar high affinities for aFGF and bFGF (Dionne et al., 1990; Crumley et at., 1991).Thus, Ig domain I of FGFR 2 does not appear to be necessary for high affinity binding of aFGF and bFGF. Crumley et al. (1991) have reported the isolation of an FGFR 2 cDNA that encodes a signal peptide, a single Ig-like domain, an acid box domain, and a stop codon. Comparison of this protein to the secreted Ig domain I form of FGFR 1 (Fig. 5, i) indicates that these two proteins are structurally related, but not identical.

B. VARIATIONSINVOLVING THE ACIDBox DOMAIN

To identify cDNA clones for the KGF receptor Miki et al. (1991) employed a novel expression cloning strategy. The approach that was taken involved transfecting 3T3 cells expressing KGF, but not the KGF receptor, with cDNAs derived from cells that express the receptor. Transformed foci that were obtained were then studied in further detail. Cells from one of the transformed foci were found to express high affinity binding sites for KGF. This led to the isolation of a KGF receptor (KGFR) cDNA. Interestingly, the cDNA encoding the KGF receptor represents a unique splice variant of the FGFR 2 gene (Fig. 5, vii). The protein predicted by this cDNA is missing both Ig domain I and the acid box domain, and contains sequences corresponding to the IIIb exon in the third Ig domain. This represented the first example of an FGF receptor protein that was missing the acid box domain. In the FGFR 1 gene, the acid box domain is encoded by a single distinct exon (Fig. 6). It remains to be seen, however, whether alternatively spliced mRNAs that code for FGFR 1 proteins without the acid box domain exist. The acid box represents a peculiar hallmark feature of the FGF receptors. At present, though, the function of this domain remains unclear. Since the KGF receptor also contained sequences in the third Ig domain different from sequences in other FGFR 2 proteins (Fig. 5 , ix and x) whose binding properties were known to be different, the differences in ligand binding specificities between these receptors could not be attributed solely to the presence or absence of the acid box domain.

FGF RECEPTOR MULTIGENE FAMILY

21

In the case of FGFR 1, however, deletion of this domain from 3 Ig or 2 Ig receptor forms does not affect the affinity of these receptors for either aFGF or bFGF (de Vries and Williams, 1992).

C. ALTERNATIVE EXONS FOR I11 HALFOF Ig DOMAIN

THE

SECOND

Several groups have reported the isolation of cDNAs encoding FGFR 2 proteins with different amino acid sequences in the second half of the Ig domain 111. Some of these cDNAs contain sequences corresponding to the IIIb exon of FGFR 1 [Hattori et al. (K-Sam), 1990; ChampionArnaud et al. (K-Sam’), 1991; Miki et al. (KGFR), 1991; Sat0 et al., 1991; Dell and Williams, 19921, whereas the others contain sequences corresponding to the IIIc exon of FGFR 1 [Dionne et al. (BEK), 1990; Houssaint et al. (TK14), 1990; Pasquale (CekS), 1990; Champion-Arnaud et al. (TK25), 1991; Raz et al., 19911. No FGFR 2 cDNAs that contain sequences corresponding to the IIIa (secreted form) exon of FGFR 1 have been reported. Thus, at a minimum there are at least two alternative exons for the second half of Ig domain I11 in FGFR 2. Analyses of the region of the human FGFR 2 gene encoding Ig domain I11 have revealed that the FGFR 2 gene is organized in a fashion nearly identical to that of the FGFR 1 gene (Champion-Arnaud et al., 1991;Johnson et al., 1991). The linear arrangement of the IIIb and IIIc exons in the FGFR 1 and FGFR 2 genes is identical, and the positions and sizes of intron sequences are also similar. In contrast, the putative IIIa exon of the FGFR 2 gene (based on corresponding location to that of the FGFR 1 IIIa exon) would contain an open reading frame that codes for only four amino acids (Johnson et al., 1991). Thus, a putative FGFR 2 (IIIa) secreted form would be considerably shorter than the FGFR 1 (IIIa) secreted form. More studies are needed to determine whether an authentic FGFR 2 IIIa exon is present in the FGFR 2 gene and whether this exon is expressed in vivo. Figure 7 shows a comparison of the IIIb and IIIc sequences of FGFR 1 and FGFR 2. It is interesting to note that the FGFR 1 IIIb sequence is more closely related to the FGFR 2 IIIb sequence (78% identity) than to the FGFR 1 IIIc sequence (45%). Also, the FGFR 1 IIIc sequence is more closely related to the FGFR 2 IIIc sequence (83%) than to the FGFR 1 IIIb sequence (45%). The FGFR 2 IIIb and IIIc sequences exhibit 51% identity. These numbers demonstrate that there is greater divergence between similar exons of the same gene than between corresponding exons of different genes. This suggests that the existence of the IIIb and IIIc exons is primordial to the existence of a multigene family.

22

DANIEL E. JOHNSON AND LEWIS T. WILLIAMS

A

LL.U

LLLC

IUh ~~

I..I.,k

-HSGINSSDAE--VLTLFNVTEAQSGEYVCKVSNYIGEANQSAWLTVTRP I I I I II I II I l l 1 I l l IIIIII -TAGVNTTDKEMEVLHLRNVSFEDAGEYTCLAGNSIGLSHHSAWLTVLE

45

HSGINSSNAE--VLALFNVTEADAGEYICKVSNYIGQANQSAWLTVLPK00 __ I I I II Ill I I I I I I I l l IIIIIIII AAGVNTTDKEIEVLYIRNVTFEDAGEYTCLAGNSIGISFHSAWLTVLP

51 %

%

C FGFR 1 Luh

U

HSGINSSDAEVLTLFNVTEAQSGEYVCKVSNYIGEANQSAWLTVTRP IIIIIII IIII IIIIIII I l l IIIIIIII IIIIIIII1 HSGINSSNAEVLALFNIPPEADAGEYICKVSNYIGQANQSAWLTVLPKQQ

78

%

D k X J 3 X m TAGVNTTDKEHEVLHLRNVSFEDAGEYTCLAGNSIGLSHHSAWLTVLE IIIIIIIII Ill Ill 111lI1IIIIlIIIII I OIIIIII FGFR 2 IIIC AAGVNTTDKEIEVLYIRNVTFEDAGEYTCLAGNSIGISFHSAWLTVLP

83 %

FIG. 7. Comparison of the IIIb and IIIc amino acid sequences of human FGFR 1 and human FGFR 2. The figure shows (A) an alignment of the FGFR 1 IIIb and FGFR 1 IIIc sequences; (b) an alignment of the FGFR 2 IIIb and FGFR 2 IIIc sequences; (C) an alignment of the FGFR 1 IIIb and FGFR 2 IIIb sequences; and (D) an alignment of the FGFR 1 IIIc and FGFR 2 IIIc sequences. The numbers on the right indicate the degree of amino acid identity between the two sequences. The FGFR 1 IIIb sequence was derived from sequences in the human FGFR 1 gene Uohnson el al., 1991). The remaining sequences were derived from cDNA sequences discussed in the text.

D. THEINCLUSIONOR EXCLUSION OF Two AMINO ACIDSIN THE JUXTAMEMBRANE DOMAIN The isolation of FGFR 2 cDNAs encoding proteins containing (or missing) two additional amino acids in the juxtamembrane region has been reported by several groups (Hattori et al., 1990; Houssaint et al., 1990; Champion-Arnaud et al., 1991). In the human FGFR 2 sequence shown in Fig. 3 these amino acids correspond to T h r (429) and Val (430). The location of this dipeptide corresponds precisely to the location of an

FGF RECEPTOR MULTIGENE FAMILY

23

exonhntron boundary in the FGFR 1 gene. It remains to be seen whether the presence or absence of this dipeptide results from the use of slightly different splice donor sites.

E. THREE ALTERNATIVE EXONSFOR THE C-TAILDOMAIN The FGFR 2 sequence shown in Fig. 3 (also Fig. 5, ix) contains a C-tail domain of 61 amino acids. Two additional cDNA clones have been isolated from human tumor cDNA libraries that encode two distinct C-tail domains [Fig. 5 , viii (K-Sam)and Fig. 5, xi (TK25); Hattori et al., 1990; Champion-Arnaud et al., 19911. The two distinct C-tail domains both diverge from the FGFR 2 (Fig. 3) sequence following amino acid 760 (Ile). The sequence of one of these C-tail domains [Fig. 5, viii (K-sam)]is only 12 amino acids long and is highly divergent from the C-tail sequences of other members of this family (Fig. 5). The sequence of the other C-tail domain [Fig. 5, xi (TK25)] is 27 amino acids long and bears considerable homology to a portion of the C-tail sequence found in other FGFR 2 proteins. It remains to be determined whether these shorter, variant C-tail domains are expressed in normal cells. This is an important point, since previous studies have shown that mutations in the C-tail domains of receptor tyrosine kinases can lead to increased transforming potential of these proteins (Ullrich and Schlessinger, 1990).

X. FGFR 3 and FGFR 4 Currently, only single cDNAs have been isolated for FGFR 3 and FGFR 4 (Fig. 3 and Fig. 5, xii and xiii). In each case, the cloned cDNAs encode 3 Ig domain receptor forms containing IIIc type sequences in the third Ig domain (Keegan et al., 1991; Partanen et al., 1991). The FGFR 4 protein is somewhat unique in that it contains a core of only four consecutive (or five of seven) acidic residues in the acid box domain (see Fig. 3). This is somewhat shorter than the core sequence found in the acid box domains of FGFR 1 (eight consecutive acidic residues), FGFR 2 (six of seven acidic residues), or FGFR 3 (seven of eight acidic residues). In general, though, the structures of FGFR 3 and FGFR 4 closely resemble those of FGFR 1 and FGFR 2. In the future it will be interesting to determine whether multiple forms of FGFR 3 and FGFR 4 mRNA transcripts also exist.

24

DANIEL E. JOHNSON AND LEWIS T. WILLIAMS

XI. Ligand Binding Specificities of the Cloned FGF Receptors

Prior to the cloning of FGF receptor cDNAs, it was unclear whether multiple members of the FGF family could bind to a common receptor, and whether multiple forms of the receptor, if they existed, would exhibit different ligand binding specificities. Binding studies on cells transfected with cloned FGF receptor cDNAs have now provided conclusive answers to these questions. Initial studies demonstrated that 3 Ig and 2 Ig FGFR 1 forms containing IIIc-type sequences (Fig. 5, iv and v) bind either aFGF (Kd = 20-80 pM) or bFGF (Kd = 50-150 pM) with high affinity (Dionne et al., 1990; Johnson et al., 1990). Both ligands also activate receptor tyrosine kinase activity and receptor-mediated signaling (Dionne et al., 1990; Johnson et al., 1990; Mansukhani et al., 1990). Additional experiments have shown that both receptor forms also bind hstlKFGF, albeit with reduced affinity compared to the binding of aFGF and bFGF (Dionne et al., 1990; Mansukhani et al., 1990). Taken together, these experiments make two important points: (a) Multiple members of the FGF family can bind to the same receptor species and ( 6 ) the first Ig domain (I) of FGFR 1 is not essential for high affinity binding of aFGF or bFGF. Similar conclusions can also be drawn from studies of FGFR 2. In this case, a 3 Ig FGFR 2 form containing IIIc-type sequences (we will use the following nomenclature to refer to this receptor: 3 Ig/IIIc/FGFR 2; see Fig. 5, ix) has been shown to bind and become activated by aFGF (Kd = 40-100 pM), bFGF (Kd = 80-150 pM), and hstlKFGF (Dionne et al., 1990). Acidic and basic FGF also stimulate FGFR 3 activation (Keegan et al., 1991). Thus, as with FGFR 1, multiple members of the FGF family bind and activate both FGFR 2 and FGFR 3 proteins. In view of the fact that multiple FGF receptor proteins can bind multiple FGFs, how then could cells o r tissues selectively respond to individual members of the FGF family? Selective responsiveness to individual FGFs would seem to make sense if it is important to maintain such a large family of closely related ligands. indeed, several examples of ligand-specific responsiveness have been observed. Basic FGF, but not aFGF, is reported to stimulate mitogenesis in human melanocytes (Halaban et al., 1987). Also, aFGF and bFGF stimulate the proliferation of fibroblast and endothelial cells, whereas KGF does not (Rubin e l al., 1989).Thus, there must be mechanisms for achieving selective responsiveness to different members of the FGF family. One possibility is that variant receptor forms derived from the same gene by alternative splicing could exhibit different Iigand binding characteristics. Tissue-specific

FGF RECEPTOR MULTIGENE FAMILY

25

responsiveness to different FGFs could then be achieved by tissue-specific alternative splicing. Another possibility is that analogous splice variants derived from different FGF receptor genes encode receptor proteins with different ligand binding properties. In this case selective responsiveness could be achieved by tissue-specific expression of the different genes. As it turns out, both of these possibilities appear to be true. XII. Alternative Splicing in the Third lg Domain Is Important for Determining Ligand Binding Specificities

The isolation of cDNAs encoding a receptor for KGF provided a starting point for answering questions regarding the role of alternative splicing in determining receptor-ligand interactions. The receptor encoded by the KGFR cDNA (Fig. 5, vii) binds KGF (Kd = 180-480 pM) and aFGF with equal and high affinity (Miki et al., 1991). However, bFGF is 15- to 20-fold less effective than KGF or aFGF at competing with the binding of iodinated KGF to this receptor (Bottaro et al., 1990; Miki et al., 1991). These results differ from those obtained with a 3 Ig form of FGFR 2 containing IIIc sequences (3 Ig/IIIc/FGFR 2; Fig. 5, ix) (Dionne et al., 1990). This receptor protein binds aFGF (Kd = 40-100 pM) and bFGF (Kd = 80-150 pM) with high affinity, but presumably does not bind KGF (since fibroblasts that express this receptor are nonresponsive to KGF). These two receptor species differ in three regions: (a) KGFR is missing Ig domain I; (b) KGFR is missing the acid box domain; and (c) KGFR contains IIIb-type sequences in the third Ig domain, whereas the 3 Ig/IIIc/FGFR 2 form contains IIIc-type sequences. Hence, the different binding specificities of these proteins must be due to one or more of these three domains. Experiments with FGFR 1 proteins have helped to clarify the importance of the aforementioned three domains for ligand binding. As previously discussed (Section XI), both the 3 Ig and the 2 Ig forms of FGFR 1 bind aFGF and bFGF with nearly equal affinities (Dionne et al., 1990; Johnson et al., 1990). Thus Ig domain I is not essential for high affinity binding of these two ligands. Likewise, removal, via mutagenesis, of the acid box domain from either receptor form does not affect binding affinities for aFGF and bFGF (de Vries and Williams, 1992). These results suggested that the reduced affinity of the KGF receptor for bFGF may be due the presence of IIIb (as opposed to IIIc) sequences in this receptor form. To examine the functional importance of the second half

26

DANIEL E. JOHNSON AND LEWIS T. WILLIAMS

of Ig domain 111, Werner et al. (1992a) replaced IIIc sequences in the 2 Ig/IIIc/FGFR 1 (Fig. 5 , v) protein with IIIb sequences. T h e resulting 2 Ig/IIIb/FGFR 1 protein bound aFGF with high affinity, but had a much weaker affinity for bFGF. Thus, binding specificity for bFGF can be determined on the basis of which exon (IIIb or IIIc) is used to code for the second half of Ig domain I. Further experiments are needed to determine whether the 2 Ig/IIIb/FGFR 1 protein will also bind KGF. A summary of binding data for FGFR 1 and FGFR 2 forms containing different sequences in the third Ig domain is shown in Table 111. In a further series of experiments, Duan et al. (1992) have shown that a secreted FGFR 1 form containing IIIa sequences in the third Ig domain binds bFGF with higher affinity than aFGF. This leads to the conclusion that alternative splicing in the third Ig domain also is important for determining binding specificity for aFGF. Additional convincing evidence regarding the importance of alternative splicing in the third Ig domain has been demonstrated for the FGFR 2 proteins. Crumley et al. (1991) have recently isolated a cDNA encoding an FGFR 2 protein that is identical to the KGF receptor (Fig. 5 , vii) except that it contains IIIc-type sequences instead of IIIb-type sequences in the third Ig domain. This protein binds both aFGF and bFGF with high affinity. Thus, from these experiments, it can be concluded that alternative splicing in the third Ig domain of FGFR 2 is important for determining binding specificity for bFGF. In a related study, Dell and Williams (1992) have isolated a cDNA encoding an FGFR 2 protein that TABLE 111 BINDING OF FGFs TO FGFR 1 A N D FGFR 2 FORMS CONTAINING DIFFERENT SEQUENCES IN THE THIRD Ig DOMAIN (111) FGFR 1 IIIa IIIb IIIC

bFGF > aFGF aFGF > bFGF aFGF = bFGF

FGFR 2 IIIb IIIC

aFGF = KGF > bFGF aFGF = bFGF (KGF does not bind)

Nofolu. This fable s h o w the relative affinities of different FGF receptor forms for aFGF, bFGF, and KGF. The depicted receptors differ in the sequences of their third Ig domains, containing either IIla. Illb, or IIlc sequences. The following sources were used to compile this figure: Dionne el al. (1990); Johnson ef al. (1990): Mansukhani et 01. (1990);kliki d al. (1991);Crumley et al. (1991); Werner et af. (l992a); and Dell and Williams (1992).

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is identical to 3 Ig/IIIc/FGFR 2 (Fig. 5, ix) except that it contains IIIbtype sequences. This protein binds aFGF with high affinity but exhibits only low affinity binding for bFGF. The findings presented above can be summarized as follows (see Table 111): (a) Acidic FGF binds with high affinity to FGFR 1 and FGFR 2 forms containing either IIIb or IIIc sequences regardless of whether Ig domain I and the acid box domain are also present. In contrast, aFGF binds with only low affinity to a 2 Ig FGFR 1 form containing IIIa sequences. (b) Basic FGF binds with high affinity to FGFR 1 and FGFR 2 forms containing IIIc sequences regardless of the presence or absence of Ig domain I and the acid box domain. Also, bFGF binds to the 2 Ig FGFR 1 form containing IIIa sequences. In contrast, bFGF binds with only low affinity to FGFR 1 and FGFR 2 forms that contain IIIb-type sequences. (c) KGF binds with high affinity to an FGFR 2 form that is missing Ig domain I and the acid box domain, and contains IIIb type sequences. In contrast, KGF does not bind to FGFR 1 and FGFR 2 proteins containing IIIc-type sequences. It remains to be determined whether the presence or absence of Ig domain I and the acid box domain have any additional influence on KGF binding (although one might predict that they do not). Furthermore it remains to be determined whether KGF binds to FGFR 1 forms containing either IIIa or IIIb sequences. At any rate, these binding experiments clearly demonstrate that alternative splicing in the third Ig domain is important for determining receptor binding specificities for all three FGFs that have been comparatively studied. XIII. Analogous Splice Variants from Different FGF Receptor Genes Encode Receptor Forms with Different Ligand Binding Specificities Two observations can be cited as proof that analogous receptor forms from different genes exhibit different ligand binding specificities: (a)A 3 Ig FGFR 4 form containing IIIc sequences (3 Ig/IIIc/FGFR 4; Fig. 5, xiii) binds aFGF with high affinity but does not bind bFGF (Partanen et al., 1991). Thus, the FGFR 4 receptor differs from corresponding 3 Ig/IIIc forms of FGFR 1 (Fig. 5, iv) and FGFR 2 (Fig. 5 , ix) which are known to bind aFGF and bFGF with comparable affinities (Dionne et al., 1990;Johnson et al., 1990). Since these three proteins are identical with respect to domain structure, the difference in affinities for bFGF cannot be attributed to alternative splicing and must be the result of other differences between the genes encoding these proteins. (b) It has been reported that hst/KFGF binds with high affinity to the 3 Ig/IIIc/FGFR 2 form (Fig. 5 , ix), but with reduced affinity to 3 IglIIIclFGFR 1 and 2

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Ig/IIIc/FGFR 1 forms (Fig. 5, iv and v) (Dionne et al., 1990; Mansukhani et al., 1990). This difference also cannot be attributed to alternative splicing and must be the result of additional differences between the FGFR 1 and FGFR 2 genes. XIV. Regulation of FGF Receptor Expression

The hypothesis that cells or tissues could potentially achieve selective responsiveness to different FGFs by mechanisms involving alternative splicing or preferential expression of different FGF receptor genes is well supported by the binding studies presented above. The prediction that differential expression of different FGF receptor forms and genes actually occurs in nivo has recently been verified by studies from several laboratories. These studies have revealed several examples of cell- and tissue-specific alternative splicing as well as differential expression of the different FGF receptor genes in a variety of tissues. XV. Cell- and Tissue-Specific Alternative Splicing of FGF Receptor mRNAs

A. CELL-AND TISSUE-SPECIFIC ALTERNATIVE SPLICING OF THE THIRD Ig DOMAIN In their original studies with KGF, Rubin et al. (1989) noted that keratinocytes express receptors for KGF, but fibroblasts and endothelial cells do not. In contrast, fibroblast and endothelial cells express receptors that bind bFGF. From the binding studies discussed above it is clear that KGF binds to FGFR 2 (and possibly FGFR 1) forms that contain IIIb sequences and not IIIc sequences, whereas bFGF binds to FGFR 1 and FGFR 2 forms that contain IIIc sequences and not IIIb sequences. Therefore the different binding properties of keratinocytes and fibrohlasts/endothelial cells most likely reflect differential expression of IIIb and IIIc exons in these cells. Johnson et al. (1991) have shown that several human cell lines simultaneously express transcripts containing the IIIa, IIIb, or IIIc exons of FGFR 1. In each of these cell lines, however, expression levels of the IIIc exon appear to be much higher than those of the IIIa or IIIb exons. One cell line, foreskin fibroblasts, was found to express only the IIIc exon. Werner et al. (1992a) have demonstrated differential expression of the IIIa, IIIb, and IIIc exons of FGFR 1 in mouse tissues. Whereas the IIIc exon was expressed in all tissues examined with the exception of liver, the IIIa and IIIb exons exhibit more restricted patterns of expression.

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The IIIa exon was expressed in brain, skeletal muscle, and skin. The IIIb exon was expressed predominantly in skin, and at lower levels in brain, kidney, muscle, and placenta. As was observed in studies of human cell lines, in all cases where there is simultaneous expression of more than one exon, expression levels of the IIIc exon appear to be much higher than those of the IIIa or IIIb exons.

B. TISSUE-SPECIFIC ALTERNATIVE SPLICING INVOLVING Ig DOMAIN I Although the first Ig domain of the FGFR 1 may prove unimportant for ligand binding, it is clear that removal of this domain via alternative splicing occurs in a tissue-specific fashion. Northern blotting and RNase protection assays show that 3 Ig domain forms of FGFR 1 are the predominant forms of receptor expressed during mouse embryogenesis (Reid et al., 1990; Werner et al., 1992b). In fact, 2 Ig domain forms are not detected by protection analyses until after birth. Following birth, 3 Ig and 2 Ig forms are simultaneously expressed at nearly equal levels in a number of different tissues, including heart, lung, and muscle (Werner et al., 1992b).In brain and kidney, however, 3 Ig domain forms continue to be the predominant, if not exclusive, form expressed.

XVI. Differential, Tissue-Specific Expression of the Different FGF Receptor Genes Several studies that help to document differential expression of the different FGF receptor genes in a variety of tissues have been completed (Kornbluth et al., 1988; Reid et al., 1990; Heuer et al., 1990; Wanaka et al., 1990, 1991; Sat0 et al., 1991; Stark et al., 1991; Peters et al., 1992a,b; Werner et al., 1992b). These studies have utilized the techniques of Northern blotting, RNase protection analyses, and in situ hybridization, and have used a variety of probes. It is important to note that although these studies clearly delineate expression patterns of the different FGF receptor genes, typically they do not provide information on expression of specific alternatively spliced mRNAs. In general, the FGFR 1 and FGFR 2 genes exhibit broad but distinct patterns of expression during development and in adult animals. On the other hand, the FGFR 3 and FGFR 4 genes appear to have more restricted patterns of expression. In the developing embryo, FGFR 1 transcripts are expressed predominantly in the brain and mesenchymal tissues (Heuer et al., 1990; Wanaka et al., 1990, 1991; Peters et al., 1992a),

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whereas FGFR 2 transcripts are expressed predominantly in brain and epithelium (Peters el al., 1992a; Werner et al., 1992b). FGFR 3 transcripts are expressed predominantly in brain, spinal chord, and cartilage rudiments of developing bone (Peters et al., 1992c). FGFR 4 transcripts are expressed in developing endoderm and the myotomal component of the somite, as well as myotomally derived skeletal muscle (Stark et al., 1991). N o expression of FGFR 4 transcripts was seen in cardiac muscle, however (Stark et al., 1991). In adult animals, FGFR 1 transcripts are detected in brain, bone, kidney, skin, lung, heart, and muscle, but not in liver (Heuer et al., 1990; Wanaka et al., 1990, 1991; Peters et al., 1992a; Werner et al., 1992b). FGFR 2 transcripts in adult animals are detected in brain, kidney, skin, lung, and liver, but not in heart, spleen, or muscle (Kornbluth et al., 1988; Peters et al., 1992a; Werner et al., 1992b). The pattern of expression of FGFR 1 in the central nervous system is consistent with neuronal expression, whereas the pattern of FGFR 2 expression in the CNS is more consistent with glial cell expression (Heuer et al., 1990; Wanaka et al., 1990, 1991; Peters et al., 1992a,b). FGFR 3 transcripts are detected in adult animals in brain, kidney, skin, and lung (Peters et al., 1992c).

XVII. The Drosophila FGF Receptor Glazer and Shilo ( 1 99 1) have recently reported the isolation of genomic clones for a Drosophila FGF receptor gene by low stringency screening of a Drosophila genomic library with a mouse FGFR 1 cDNA probe. The genomic clones were then used to isolate a nearly full-length cDNA clone (missing 5' nontranslated and signal peptide sequences) from a Drosophzla cDNA library. The protein encoded by this cDNA is structurally similar to the other membrane-spanning FGF receptors w e have discussed. The extracellular region of the Drosophilu FGF receptor contains 3 Ig-like domains and an acid box domain (core sequence: EDNDDDVE). The intracellular regon contains a relatively long juxtamembrane domain of approximately 90 amino acids, a tyrosine kinase domain that is interrupted by a kinase insert region of 22 amino acids, and a C-tail domain of approximately 70 amino acids. Comparison of the genomic and cDNA clones revealed that they are colinear with the exception of a single 85-bp intron in the kinase domain. The location of this intron is conserved in the human FGFR 1 gene (Johnson et al., 1991). What is striking about the genomic sequence of the Drosophila FGF receptor is the lack of introns in the extracellular coding region. This finding allows two conclusions to be drawn regarding the complexity of

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FGF receptor expression in Drosophila. First, since the coding region for the first Ig domain (I) is not separated from the remainder of the receptor gene by intron sequences, the potential for removing this domain via alternative splicing appears to be restricted to higher eukaryotes. The fact that higher eukaryotes express predominantly 3 Ig receptor forms (as opposed to 2 Ig) during development suggests that Ig domain I may have a function that is critically important during development. Experiments using Drosophila embryos may help to identify the function of this domain. Second, because the extracellular domain of the Drosophila gene does not contain introns, there is only one coding sequence for the second half of Ig domain 111. The amino acid sequence of this domain exhibits nearly equal identity with the human FGFR 1 IIIb sequence (38%) and the human FGFR 1 IIIc sequence (40%). In higher eukaryotes, receptors containing different sequences ( M a , IIIb, or IIIc) in this region exhibit distinctive ligand binding specificities (see Section XII). The existence of only one alternative for Ig domain I11 in the Drosophila FGF receptor indicates that this receptor is considerably less flexible, and suggests that the Drosophila FGF family may be less complex than that of higher eukaryotes. Glazer and Shilo (1991) also conclude that there is only one FGF receptor homolog in Drosophila based on their inability to isolate more than one class of FGFR clones. This conclusion also draws support from studies of FGF receptors from higher eukaryotes. As discussed in Section IX,C, comparison of alternative exons for Ig domain 111, both within the same gene and also between different genes, suggests that the existence of alternative exons for this receptor region is primordial to the existence of a multigene family. Since the Drosophila FGF receptor does not contain alternative exons in this region, it makes sensqto argue that there is only one FGF receptor gene in Drosophila. It seems likely, therefore, that both the FGF family and the FGF receptor family are considerably less complex in Drosophila than they are in higher eukaryotes.

XVIII. FGF Receptor-Mediated Signal Transduction Treatment of cells with FGFs leads to increased intracellular pH and intracellular Ca2+ levels (Tsuda et al., 1985; Halperin and Lobb, 1987); increased hydrolysis of polyphosphoinositides (Brown et al., 1989); increased phosphorylation of cellular proteins (Huang and Huang, 1986; Pelech et al., 1986; Coughlin et al., 1988); and increased transcription of a subset of cellular genes, including c-myc and c-fos (Kruijer et al., 1984; Muller et al., 1984; Stumpo and Blackshear, 1986). Depending on the

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cell type, exposure to FGFs ultimately leads to proliferation, differentiation, inhibition of differentiation, or maintenance of a differentiated phenotype. The signaling mechanisms that give rise to these ultimate manifestations, however, remain largely unknown. It is known that FGF-dependent signaling is initiated immediately following the binding of FGF to its receptor. The binding of aFGF or bFGF to its receptor induces receptor dimerization (Bellot et al., 1991; Ueno et al., 1992), similar to what has been observed for several other growth factor receptors (Williams, 1989; Ullrich and Schlessinger, 1990). Interestingly, both homodimeric and heterodimeric receptor species can be formed between the FGFR 1, the FGFR 2, and the FGFR 3 proteins (Bellot et at., 1991; Ueno et al., 1992). Binding also leads to activation of FGF receptor tyrosine kinase activity and receptor autophosphorylation (Huang and Huang, 1986; Coughlin et al., 1988; Mansukhani et al., 1990). Phosphorylation of dimerized receptors appears to occur via an intermolecular transphosphorylation mechanism (Bellot et al., 1991).Activation of the receptor tyrosine kinase also leads to increased tyrosine phosphorylation of a number of cellular proteins (Huang and Huang, 1986; Coughlin et al., 1988; Bottaro et al., 1990; Burgess et al., 1990; Mansukhani et al., 1990; Miki et al., 1991; Peters et al., 1992d). Phosphorylation of a 90-kDa protein (and possibly others) may be unique to FGF receptor signaling pathways (Coughlin et al., 1988). It remains unclear, however, how many (if any) of these proteins associate with and are directly phosphorylated by the FGF receptor. Proteins that are known to associate with other growth factor receptors include: Raf-1 (Morrison et al., 1989), GTPase-activating protein (GAP) (Molloy et al., 1989; Ellis et al., 1990; Kaplan et al., 1990; Kazlauskas et al., 1990), pp60c-src (Krypta et al., 1990), the p85 subunit of phosphatidylinositol 3-kinase (Coughlin et al., 1989; Bjorge et al., 1990; Kazlauskas and Cooper, 1990; Escobedo et al., 1991; Otsu et al., 1991),v-Crk (Matsuda et al., 1990; Mayer and Hanafusa, 1990), and phospholipase C-y (PLC-y) (Wahl et al., 1988, 1989; Kumijan et al., 1989; Margolis et al., 1989; Meisenhelder et al., 1989; Burgess el al., 1990; Morrison et al., 1990). Currently, only one protein, PLC-y has been identified as a candidate substrate of an FGF receptor. PLC-y is phosphorylated on tyrosine residues following FGF stimulation (Burgess et al., 1990) and direct association with the receptor has been demonstrated (Mohammadi et al., 1991; Peters et al., 1992d). Mohammadi et al. (1991) have shown that the SH2 (src homology region 2) domain of PLC-y mediates binding to the FGF receptor. In addition, a 28 amino acid peptide derived from the C-tail region of FGFR 1 has been shown to bind to the SH2 domain of PLC-y (Mohammadi et al., 1991). This peptide contained phosphorylated Tyr

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(766) (see Fig. 3). This tyrosine residue is conserved across all four FGF receptor genes. Mutation of Tyr (766) to Phe generates a receptor protein that does not associate with or phosphorylate PLC-y and does not mediate FGF-dependent phosphotidylinositol turnover (Peters et al., 1992d). The mutant receptor does, however, autophosphorylate and mediate increased tyrosine phophorylation of other cellular proteins (Peters et al., 1992d). Additional experiments with this mutant receptor should help to determine the role of PLC-y in FGF receptor-mediated signaling. Although the experiments described above have shed new light on the interaction between PLC-y and FGF receptors, the precise role of PLC-y in FGF receptor-mediated signaling remains unclear. In CCL39 cells (Chinese hamster lung fibroblasts),for instance, FGF does not stimulate hydrolysis of polyphosphoinositides (Magnaldo et al., 1986), indicating that the FGF receptors on these cells do not couple to PLC-y signaling pathways. Also, in cells where FGF does stimulate polyphosphoinositide breakdown, the magnitude of this response is much less than that observed following stimulation with other growth factors, such as PDGF (Peters et al., 1992d). Furthermore, cells expressing the Tyr (766) to Phe (766) mutant FGF receptor proliferate in response to FGF (Peters et al., 1992d), indicating that PLC-y may not be important for pathways leading to FGF-induced mitogenesis. The potential involvement of other signaling molecules in FGF receptor-mediated signaling remains largely untested. It appears likely though, that GAP protein, in contrast to its interaction with the PDGF receptor, does not associate with the FGF receptor (Molloy et al., 1989). Stimulation of cells with FGF does, however, lead to hyperphosphorylation of c-Raf-1 (Morrison et al., 1988). Currently, it is not known whether c-Raf-1 is directly phosphorylated by the receptor, or whether the phosphorylation of c-Raf-1 affects its kinase activity. Finally, although several cellular proteins are known to become phosphorylated on tyrosine residues in response to FGF treatment, the identities of these proteins (with the exception of PLC-y) remain unknown. XIX. Concluding Remarks

The FGF receptor field has grown increasingly complex in recent years with the discovery of four distinct FGFR genes. Alternative splicing of mRNA transcripts from at least two of these genes has introduced even further complexity. Binding studies discussed in this review have shown that although the multiple FGF receptor forms exhibit some common binding properties, considerable differences in binding specificities

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also exist. Functional differences between the different receptor forms were observed at two levels. First, different FGF receptor forms derived from the same gene via alternative splicing have different ligand binding properties. Specifically, the binding properties of a receptor can be determined in part by which exon (IIIa, IIIb, or IIIc) is used to code for the second half of Ig domain 111. Second, analogous splice variants from different FGF receptor genes bind different members of the FGF family. For instance, FGFR 1 and FGFR 4 forms that are identical with respect to domain structure show differences in their ability to bind bFGF. Recently, studies of FGFR mRNA localization have demonstrated tissuespecific alternative splicing of FGFR mRNA transcripts as well as tissuespecific expression of different FGFR genes. Thus, it can be concluded that mechanisms involving alternative splicing or differential expression of genes can be used to achieve selective responsiveness of different tissues to different members of the FGF family. The discovery of multiple forms of the FGF receptor raises many important questions for future investigations. It is particularly important to elucidate the roles of each of the different FGF receptor forms, particularly during development. Based on preliminary studies of FGFR mRNA localization, it seems likely that the different receptor forms have distinct roles. To determine these roles, it will be necessary to selectively knock out, by genetic or other means, specific receptor forms. Amaya et al. (199 1) and Ueno et al. (1992) have already achieved some success in knocking out FGFR function using dominant negative technology. However, it is clear that new approaches will be needed to selectively knock out individual receptor forms. Other questions that need to be addressed concern the nature of FGF receptor-mediated signal transduction. What are the signaling pathways (and molecules) used by the FGF receptors? Do the products of different FGFR genes couple to different signaling molecules and pathways? Do receptor forms that differ only in the sequences of their third Ig domain or C-tail region couple to different pathways? Do different members of the FGF family induce distinct signaling mechanisms? These and other questions need to be addressed in order to understand how FGFs can exert such a wide variety of effects in responsive cells types. Answers to these questions will provide a basis for understanding which receptor forms and which signaling pathways are involved in FGF-dependent cellular responses such as mitogenesis, chemotaxis, neurite outgrowth, neuronal maintainance, and inhibition of differentiation. Another important avenue for future research is the rational design of agonists and antagonists of FGF actions. The design of such molecules will benefit greatly from studies of the ligand binding domains of the

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different FGF receptor forms. Initially these studies will be complicated by the fact that there are several members in the PGF family, and by the fact that there are multiple FGFR genes and multiple exons for the second half of the third Ig domain. The advantage of studying FGFFGFR interactions, however, is that ligand binding is defined by a relatively short region of amino acids. Specifically, it has been demonstrated that 2 Ig domain forms of the FGF receptor can bind aFGF, bFGF, hstlKFGF, or KGF. Thus, ligand binding requires a maximum of 2 Ig domains (227 amino acids). Furthermore, binding studies of receptor forms with different sequences in the second half of the third Ig domain show that specificity for different FGFs can be achieved by minor variation in the composition of this short region (47-48 amino acids). Mutational analyses coupled with X-ray crystallography of ligand/receptor complexes should provide meaningful information on the ligand binding domaints) of the FGF receptor. The recent publication of the threedimensional structures of aFGF and bFGF also should provide valuable information for these studies (Zhu et al., 1991). The long-range goal of designing effective agonist and antagonists of FGF action has considerable therapeutic value. Agonists of FGF might be useful for accelerating wound healing and neovascularization, and maintaining viability of certain neuronal populations. Antagonists of FGF, on the other hand, might be useful for blocking angiogenesis in pathological conditions such as diabetic retinopathy and tumor neovascularization. ACKNOWLEDGMENTS We are deeply indebted to many colleagues for helpful insights and suggestions, and for providing data prior to publication. Without their cooperation and generosity this review would not have been possible. We thank Kevin Peters, Dah-Shuhn R. Duan, Sabine Werner, Carlie de Vries, Pauline Lee, Hikaru Ueno, Khoi Le, Dan Mirda, and Michael Jaye. This work was supported by the National Institutes of Health Program of Excellence in Molecular Biology (HL43821). D.E.J. was supported by American Heart Association Fellowship 94-1219116.

REFERENCES Abraham, J. A., Mergia, A., Whang, J. L., Tumolo, A., Friedman, J.. Hjerrild, K. A., Gospodarowicz, D., and Fiddes, J. C. (1986a). Science 438, 545-548. Abraham, J. A., Whang, J. L., Tumolo, A., Mergia, A., Friedman, J., Gospodarowicz, D., and Fiddes, J. (1986b). EMBOJ. 5, 2523-2528. Amaya, E., Musci, T. J., and Kirschner, M. W. (1991). Cell (Cambridge, Marc.)66,257-270. Anderson, K. J., Dam, D., Lee, S., and Cotman, C. W. (1988). Nature (London) 334, 360361.

36

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Armelin, H. A. (I973). Proc. Natl. Acad. Sci. U.S . A, 70,2702-2706. Baird, A., Esch, F., Ling, N., and Gospodarowicz, D. (1985a). Regul. Pept. 12, 201-213. Baird, A., Esch, F., Gospodarowicz, D., and Guillemin, R. (1985b).B i o c h i r t l y 24,78557859. Baird, A., Esch, F., Mormede, P., Ueno, N., Ling, N., Bohlen, P., Ying, S.-Y., Wehrenberg, W. B., and Guillemin, R. (1986). Recent Prog. H u m . Res. 42, 143-205. Bashkin, P., Doctrow, S., Klagsbrun, M., Svahn, C. M., Folkman, J., and Vlodavsky, I. S t 1737-1 ~ 743. (1989). B i ~ ~ h i 28, Bellot, F., Crumley, G., Kaplow,J. M., Schlessinger, J.. Jaye, M., and Dionne, C. A. (1991). EMBO J. 10,2849-2854. Bjorge, J. D., Chan, T.-O., Antczak, M., Kung, H.-J., and Fujita, D. J. (1990). Proc. Natl. Acid. Sn'. U . S.A . 87,3816-3820. Bottaro, D. P., Rubin, J. S., Ron, D., Finch, P. W., Florio, C., and Aaronson, S. A. (199O).J. Biol. C h . 465, 12767-12770. Bovi, P. D., Curatola, A. M., Kern, F. G., Greco, A., Ittmann, M., and Basilico, C. (1987). Cell (Canbridge, Mass.) 50, 729-737. Broad, T. E., and Ham, R. G. (1983). Eur. J . Biochem. 135,33-39. Brown, K. D., Blakeley, D. M., and Brigstock, D. R. (1989). FEES Lett. 247,227-231. Burgess, W. H., and Maciag, T. (1989). Annu. Rev. Biochem. 58, 575-606. Burgess, W. H., Dionne, C. A., Kaplow, J.. Mudd, R., Friesel, R., Zilbertstein, A,, Schlessinger, J., and Jaye, M. (1990). Mol. CeU. Biol. 10, 4770-4777. Burrus, L. W., and Olwin, B. B. (1989).J. Biol. Chem. 264, 18647-18653. Champion-Arnaud, P., Ronsin, C., Gilbert, E., Gesnes, M. C., Houssaint, E., and Breathnach, R. (1991). Oncogene 6, 979-987. Clegg, C. H., Linkhart, T. A., Olwin, B. B., and Hauschka, S. D. (1987).J. Cell Biol. 105, 949-956. Coughlin, S. R., Barr, P. J., Cousens, L. S., Fretto, L. J., and Williams, L. T. (1988).J. Biol. C h . 263,988-993. Coughlin, S. R., Escobedo, J. A., and Williams, L. T. (1989). Science 243, 1191-1 194. Courty, J., Dauchel, M. C., Mereau, A., Badet, J., and Barritault, D. (1988).J. Biol. C h a . 263, 11217-11220. Coussens, L., Yang-Feng, T. L., Liao, Y.-C., Chen, E., Gray, A., McGrath, J., Seeburg, P. H., Libermann, T. A., Schlessinger, J., Francke, U., Levinson, A., and Ullrich, A. (1985). Science 230, 1 132-1 139. Coussens, L., Van Beveren, C., Smith, D., Chen, E., Mitchell, R. L., Isacke, C., Verma, I. M.,and Ullrich, A. (1986). Nature (London) 340,277-280. Crumley, G., Bellot, F., Kaplow, J. M., Schlessinger, J., Jaye, M.,and Dionne, C. A. (1991). OnCogm 6,2255-2262. Cunningham, B. A., Hemperly, J. J., Murray, B. A., Prediger, E. A., Brackenbury, R.,and Edelman, G. M. (1987). Nature (London) 236, 799-806. Dell, K. R., and Williams, L. T. (1992). Submitted for publication. de Vries, C., and Williams, L. T. (1992). In preparation. Dickson, C., Smith, R., Brookes, S., and Peters, G. (1984). Cell (Cambridge, Mass.)37, 529536. Dionne, C. A., Crumley, G., Bellot, F., Kaplow, J. M., Searfross, G., Rum, M., Burgess, W. H., Jaye, M., and Schlessinger, J. (1990). EMBOJ. 9, 2685-2692. Duan, D.-S. R., Werner, S., and Williams, L. T. (1992).J. Biol. Chem. In press. Eisemann, A., Ahn, J. A., Graziani, G., Tronick, S. R., and Ron, D. (1991). Oncogene 6, 1195-1202. Ellis, C., Moran, M., McCormick, F., and Pawson, T. (1990). Nature (London) 343,377-381.

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37

Esch, F., Baird, A., Ling, N., Ueno, N., Hill, F., Denoroy, L., Klepper, R., Gospodarowicz, D., Bohlen, P., and Guillemin, R. (1985).Proc. Natl. Acad. Sci. U.S.A. 82,6507-651 1. Escobedo, J. A., Navankasattusas, S., Kavanaugh, W. M., Milfay, D., Fried, V. A., and Williams, L. T. (1991).Cell (Cambridge, Mass.)65, 75-82. Finch, P. W., Rubin, J. S., Miki, T., Ron, D., and Aaronson, S. A. (1989).Science 245,752-

755.

Folkman, J., and Klagsbrun, M. (1987).Science 235, 442-447. Friesel, R., Burgess, W. H., Mehlman, T., and Maciag, T. (1986).J.Biol. C h . 261, 7581-

7584.

Gimenez-Gallego, G., Rodkey, J.. Bennett, C., Rios-Candelore, M., DiSalvo, J., and Thomas, K. A. (1985).Science 230, 1385-1388. Glazer, L., and Shilo, B.-Z. (1991).Genes Den 5, 697-705. Gospodarowicz, D. (1974).Nature (London) 249, 123-127. Gospodarowicz, D.(1975).J. Biol. Chem. 250, 2515-2520. Gospodarowic, D., Jones, K. L., and Sato, G. (1974).Proc. Natl. Acad. Sci. U . S.A. 71,2295-

2299.

Gospodarowicz, D., Bialicki, H., and Greenberg, G. (197QJ.Biol. Chem. 253,3736-3743. Gospodarowicz, D., Cheng, J., Lui, G.-M., Baird, A., Esch, F., and Bohlen, P. (1985a). Endocrinology, (Baltimore) 117,2283-2291. Gospodarowicz, D., Cheng, J., Lui, G.-M., Fujii, D. K., Baird, A., and Bohlen, P. (1985b). Biochem. Biophys. Res. C a m u n . 30, 554-562. Gospodarowicz, D.,Baird, A., Cheng, J., Lui, G.-M., Esch, F., and Bohlen, P. (1986a). Endocrinology (Baltimore) 118, 82-90. Gospodarowicz, D., Neufeld, G., and Schweigerer, L. (1986b).Cell Dzffm. 19, 1-17. Gross, J. L., Moscatelli, D., Jaffe, E. A., and Rifkin, D. B. (1982).J.Cell Bid. 94,974-981. Gross, J. L., Moscatelli, D., and Rifkin, D. B. (1983).Proc. Natl. Acad. Sci. U . S. A. 80,2623-

2627.

Halaban, R., Ghosh, S., and Baird, A. (1987).In Vitro Cell. Den B i d . 23,47-52. Halperin, J. A., and Lobb, R. R. (1987).B i o c h . Biophy. Res. Commun. 144, 115-122. Hanks, S. K., Quinn, A. M., and Hunter, T. (1988).Science 241, 42-52. Hattori, Y.,Odagiri, H., Nakatani, H., Miyagawa, K., Naito, K., Sakamoto, H., Katoh, O., Yoshida, T., Sugimura, T., and Terada, M. (1990).Proc. Natl. Acad. Sci. U . S. A. 87,

5983-5987.

Hauschka, P.V., Mavrakos, A. E., Iafrati, M. D., Doleman, S. E., and Klagsbrun, M. (1986). J . Biol. C h a . 261, 12665-12674. Heuer, J.,von Bartheld, C., Kinoshita, Y., Evers, P., and Bothwell, M. A. (1990).Neuron 5,

283-296.

Hoffman, R. S. (1940).Growth 4,361-376. Hou, J., Kan, M., McKeehan, K., McBride, G., Adam, P., and McKeehan, W. L. (1991). Scierue 251,665-668. Houssaint, E., Blanquet, P. R., Champion-Arnaud, P., Gesnel, M. C., Torriglia, A., Courtois, Y., and Breathnach, R. (1990).Proc. Natl. Acad. Sci. U . S.A. 87,8180-8184. Huang, S. S., and Huang, J. S. (1986).J.Biol. C h a . 261, 9568-9571. Imamura, T., Tokita, Y., and Mitsui, Y. (1988).Biochem. Bwphys. Res. Commun. 155,583-

590.

Jaye, M.,Howk, R.,Burgess, W. H., Ricca, G. A., Chiu, I.-M., Ravera, M. W., OBrien, S. J., Modi, W. S., Maciag, T., and Drohan, W. N. (1986).S a k e 233, 543-545. Jaye, M., Lyall, R. M., Mudd, R., Schlessinger, J,, and Sarver, N. (1988).EMBOJ. 7,963-

969.

Johnson, D. E., Lee, P. L., Lu, J., and Williams, L. T. (1990).Mol. Cell. Biol. 10,4728-4736.

38

DANIEL E. JOHNSON AND LEWIS T. WILLIAMS

Johnson, D. E., Lu, J., Chen, H., Werner, S., and Williams, L. T. (1991).Mol. Cell. Biol. 11, 4627-4634. Kaplan, D. R., Morrison, D. K., Wong, G., McCormick, F., and Williams, L. T. (1990). Cell (Cambndge, Mars.) 61, 125-133. Kazlauskas, A., and Cooper, J. A. (1990). E M B O J . 9,3279-3286. Kazlauskas, A,, Ellis, C., Pawson, T., and Cooper, J. A. (1990). Science 247, 1578-1581. Keegan, K., Johnson, D. E., Williams, L. T., and Hayman, M. J. (1991).Proc. Natl. Acad. Sci. C'. S . A. 88, 1095-1099. Kiefer, M. C., Stephans, J. C., Crawford, K., Okino, K., and Barr, P. J. (1990). Proc. Nutl. Acad. Sci. 1'. S. A. 87,6985-6989. Kimelman, D., and Kirschner, M. (1987). Cell (Cambndge, Mass.)51, 869-877. Kimelman, D., Abraham, J. A., Haaparanta, T., Palisi, T. M., and Kirschner, M. W. (1988). S c k u e 242, 1053-1056. Klagsbrun, M., and Baird, A. (1991). Cell (Cambrulge, Mass.) 67,229-231. Klagsbrun, M., and Shing, Y. (1985). Proc. Natl. Acad. Sci. U . S. A. 82, 805-809. Kornbluth, S., Paulson, K. E., and Hanafusa, H. (1988). Mol. Cell. Biol. 8, 5541-5544. Kruijer, W., Cooper, J. A., Hunter, T., and Verma, I. M. (1984). Nature(London) 312,711716. Krypra, R. M., Goldberg, Y., Uiug, E. T., and Coutneidge, S. A. (1990). Cell (Cambridge, Mass.) 62, 481-492. Kumijan, D. A., Wahl, M. I . , Rhee, S. G., and Daniel, T. 0.(1989).Proc. Natl. Acad. Sci. U . S . A. 86, 8232-8239. Kurokawa, T., Sasada. R., Iwane, M., and lgarashi, K. (1987). FEBS Lett. 213, 189-194. Larhrop, B., Olson, E., and Glaser, L. (1985).J. Cell. Biol. 100, 1540-1547. Lee, P. L., Johnson, D. E., Cousens, L. S., Fried, V. A., and Williams, L. T. (1989). Science 245, 57-60. Libermann, T. A., Friesel, R., Jaye, M., Lyall, R., Westermark, B., Drohan, W., Schmidt, A,, Maciag, T., and Schlessinger, J. (1987). E M B O J . 6, 1627-1632. Linkhart, T. A., Clegg, C. H., and Hauschka, S. D. (1981). Deu. Biol. 86, 19-30. Lobb, R. R., Harper, J. W., and Fett, J. W. (1986). Anal. B i o c h . 154, 1-14. Maciag, T., Mehlman, T., Friesel, R., and Schreiber, A. B. (1984). Science 225, 932-934. Magnaldo, I., L'Allemain, G., Chambard, J. C., Moenner, M., Barritault, D., and Pouyssegur, J. (1986).J. Bwl. C h . 261, 16916-16922. Mansukhani, A., Moscatelli, D., Talarico, D., Levytska, V., and Basilico, C. (1990). Proc. N d l . A d . Sci. U . S. A. 87,4378-4382. Margolis, B., Rhee, S. G., Felder, S., Mervic, M., Lyall, R., Levitzki, A., Ullrich, A,, Zilbertstein, A,, and Schlessinger, J. (1989). Cell (Cambridge, Mass.) 57, 1101-1107. Marics, I., Adelaide, J., Raybaud, F., Mattei, M., Coulier, F., Planche, J., Lapeyriere, O., and Birnbaum, D. (1989). Oncogene 4, 335-340. Matsuda, M., Mayer, B. J., Fukui, Y., and Hanafusa, H. (1990). Science 248, 1537-1539. Mayer, B. J., and Hanafusa, H. (1990). Proc. Natl. Acad. Sci. U . S . A. 87, 2638-2642. Meisenhelder, J., Suh, P.-G., Rhee, S. G., and Hunter, T. (1989). Cell (Cambridge, Mass.) 57, 1109-1122. Mergia, A., Eddy, R., Abraham, J. A., Fiddes, J. C., and Shows, T. B. (1986). Biochem. Biophys. Res. Commun. 138, 644-65 1 . Mignatti, P., Tsuboi, R., Robbins, E., and Rifkin, D. B. (1989).J.Cell Biol. 108, 671-682. Miki, T., Fleming, T. P., Bottaro, D. P., Rubin, J. S., Ron, D., and Aaronson, S. A. (1991). Science 251,72-75. Moenner, M., Chevallier, B., Badet, J., and Barritault, D. (1986).Proc. Natl. Acad. Sci. U . S . A . 83, 5024-5028.

FGF RECEPTOR MULTIGENE FAMILY

39

Mohammadi, M., Honegger, A. M., Rotin, D., Fischer, R., Bellot, F., Li, W., Dionne, C. A,, Jaye, M., Rubinstein, M., and Schlessinger, J. (1991). Mol. Cell. Biol. 11, 5068-5078. Molloy, C. J., Bottaro, D. P., Fleming, T. P., Marshal, M. S., Gibbs, J. B., and Aaronson, S. A. (1989). Nature (London) 342, 711-714. Moore, R., Casey, G., Brookes, S., Dixon, M., Peters, G., and Dickson, C. (1986).EMBOJ. 5, 919-924. Morrison, D. K., Kaplan, D. R., Rapp, U., and Roberts, T. M. (1988).Proc. Nutl. Acad. Sci. U. S. A. 85,8855-8859. Morrison, D. K., Kaplan, D. R., Escobedo, J. A., Rapp, U. R., Roberts, T. M., and Williams, L. T. (1989). Cell (Cambridge, Mass.) 58,649-657. Morrison, D. K., Kaplan, D. R., Rhee, S. G., and Williams, L. T. (1990).Mol. Cell. Biol. 10, 2359-2366. Morrison, R. S., Sharma, A., De Vellis, J., and Bradshaw, R. A. (1986).Proc. Nutl. Acad. Sci. U . S. A. 83, 7537-7541. Moscatelli, D. (1987).J. Cell. Physiol. 131, 123-130. Moscatelli, D. (1988).J. Cell Biol. 107, 753-759. Moscatelli, D., and Quarto, N. (1989).J. Cell Biol. 109, 2519-2527. Moscatelli, D., Presta, M., and Rifkin, D. B. (1986).Proc. Nutl. Acad. Sci. U. S. A. 83,20912095. Muller, R., Bravo, R., Burckhardt, J., and Curran, T. (1984). Nature (London) 312, 716720. Musci, T. J., Amaya, E., and Kirschner, M. W. (1990). Proc. Nutl. Acad. Sci. U. S. A. 87, 8365-8369. Neufeld, G., and Gospodarowicz, D. (1985).J. Biol. Chem. 260, 13860-13868. Neufeld, G., and Gospodarowicz, D. (1986).J. Biol. Chem. 261, 5631-5637. Neufeld, G., Mitchell, R., Ponte, P., and Gospodarowicz, D. (1988).J. CellBiol. 106, 13851394. Nishi, N., Matuo, Y., Mugurama, Y., Yoshitake, Y., Nishikawa, K.,and Wada, F. (1985). Biochem. Biophys. Res. Commun. 132, 1103-1 109. Olwin, B. B., and Hauschka, S. D. (1986).Biochemistly 25, 3487-3495. Olwin, B. B., and Hauschka, S. D. (1989).J. Cell. Biochem. 39,443-454. Otsu, M., Hiles, I., Gout, I., Fry, M. J., Ruiz-Larrea, F., Panayotou, G., Thompson, A., Dhand, R., Hsuan, J., Totty, N., Smith, A. D., Moryan, S. J., Courtneidge, S. A., Parker, P. J., and Waterfield, M. D. (1991). Cell (Cambridge, Mars.) 65, 91-104. Partanen, J., Makela, T. P., Eerola, E., Korhonen, J., Hirvonen, H., Claesson-Welsh, L., and Alitalo, K. (1991). EMBOJ. 10, 1347-1354. Pasquale, E. B. (1990). Proc. Natl. Acad. Sci. U. S. A. 87, 5812-5816. Pasquale, E. B., and Singer, S. J. (1989). Proc. Natl. Acad. Sci. U . S. A. 86, 5449-5453. Pelech, S. L., Olwin, B. B., and Krebs, E. G. (1986).Proc. Natl. Acad. Sci. U . S. A. 83,59685972. Peters, G., Brookes, S., Smith, R., and Dickson, C. (1983). Cell (Cambridge, Mas.) 33,369377. Peters, K. G., Werner, S., Chen, G., and Williams, L. T. (1992a). Development (Cambridge, UK) 114,233-243. Peters, K. G., Basbaum, A., Williams, L. T., and Wilcox, J. (1992b).In preparation. Peters, K. G., Ornitz, D., Werner, S., and Williams, L. T. (1992~).In preparation. Peters, K. G., Marie, J., Escobedo, J., Del Rosario, M., Mirda, D., and Williams, L. T. (1992d). Submitted for publication. Presta, M., Mignatti, P., Mullins, D. E., and Moscatelli, D. (1985). Biosci. h p . 5, 783-788. Rapraeger, A. C., Krufka, A., and Olwin, B. B. (1991). Science 252, 1705-1708.

40

DANIEL E. JOHNSON A N D LEWIS T. WILLIAMS

Raz, V., Kelman, Z., Avivi, A., Neufeld. G.. Givol, D., and Yarden. Y. (1991). Oncogene 6, 753-760. Reid, H. H., Wilks, A. F., and Bernard, 0. (1990). Proc. Natl. Acad. Sci. U . S . A. 87, 15961600. Rogelj, S., Weinberg, R. A., Fanning, P., and Klagsbrun, M. (1988). Nature (London) 331, 173-1 75. Rubin, J. S., Osada, H., Finch, P. W., Taylor, W.G., Rudikoff, S., and Aaronson, S. A. (1989).Proc. Natl. Acad. Sci. U . S . A. 86,802-806. Ruta, M., Howk, R., Ricca, G., Drohan, W.. Zabelshansky, M., Laureys, G., Barton, D. E., Francke, U., Schlessinger, J., and Givol, D. (1988). Oncogene 3, 9-15. Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., Mullis, K. B., and Erlich, H. A. (1988). Science 239, 487-491. Sasada, R., Kurokawa, T., Iwane, M., and Igarashi, K. (1988). Mol. Cell. Biol. 8, 588-594. Sato, M., Kitazawa, T., Iwai, T., Seki, J., Sakato, N., Kato, J., and Takeya, T. (1991). Oncogent 6, 1279-1283. Schreiber, A. B., Kenney, J.. Kowalski, W. J., Friesel, R., Mehlman, T., and Maciag. T. (1985). PYOC.Natl. A d . Sn. U . S . A. 84,6138-6142. Serrero, G., and Khoo, J. C. (1982). Anal. B i o c h . 120, 351-359. Shing, Y.. Folkman, J.. Murray, M., and Klagsbrun, M. (1983).J. Cell B i d . 97, 395a. Shing, Y., Folkman, J., Sullivan, R., Butterfield, C., Murray, J., and Klagsbrun, M. (1984). Science 423, 1296- 1299. ~P 326, Slack,J. M., Darlington, B. G., Heath, J. K., and Godsave, S. F. (1987). N Q ~ U(London) 197-200. Stark, K. L., McMahon, J. A., and McMahon, A. P. (1991). Deuelopment (Cambrtdge, UK) 113, 64 1-65 1. S t u m p , D. J., and Blackshear, P. J. (1986). Proc. N o d Acad. Sci. Lr. S . A . 83, 9453-9457. Sullivan, R.,and Klagsbrun, M. (1985).J. Biol. C h . 260, 2399-2403. Taira, M., Yoshida, T., Miyagawa, K., Sakamoto, H., Terada, M., and Sugimura, T. (1987). Proc. Natl. Acad. Sci. U.S . A . 84,2980-2984. Thomas, K. A. (1987). FASEB 1. 1,434-440. Thomas, K. A., Rios-Candelore, M., Gimenez-Gallego, G., DiSalvo,J., Bennet, C., Rodkey, J., and Fitzpatrick, S. (1985). Proc. Natl. Acad. Sci. U . S . A. 82, 6409-6413. Togari, A., Dickens, G., Kuzuya, H., and Guroff, G. (1985).J. Neurosci. 5, 307-3 16. Trowell, 0. A., Chir, B., and Willmer, E. N. (1939).J. Exp. Biol. 16, 60-70. Tsuda, T., Kaibuchi, K., Kawahara, Y.,Fukuzaki, H., and Takai, Y. (1985). FEBS Lett. 187, 43-46. Ueno, N., Baird, A., E x h , F., Shirnasaki, S., Ling, N., and Guillemin, R. (1986). Regul. Pept. 16, 135-145. Ueno, N., Baird, A., Esch, F., Ling, N., and Guillemin, R. (1987). Mol. Cell. Endocrinol. 49, 189- 194. Ueno, H., Gunn, M., Dell, K., Tseng, A., Jr., and Williams, L. T. (1992).J. Bzol. Chem 267, 1470-1476. Ullrich, A.. and Schlessinger, J. (1990). Cell (Cambridge, Mass.) 61, 203-212. Ullrich, A., Coussens, L., Hayflick, J. S., Dull, T. J., Gray, A., Tam, A. W., Lee, J., Yarden, Y., Libermann, T. A., Schlessinger, J., Downward, J., Mayes, E. L. V., Whittle, N., Waterfield, M. D., and Seeburg, P. H. (1984). Nature (London)309,418-425. Ullrich, A., Bell, J. R., Chen, E. Y., Herrera. R., Petruzzelli. L. M., Dull, T. J., Gray, A,, Coussens, L., Liao, Y. C., Tsubokawa, M., Mason, A., Seeburg, P. H., Grunfeld, C., Rosen, 0. M., and Ramachandran, J. (1985). Nature (London) 313, 756-761. Ullrich, A.. Gray, A., Tam, A. W., Yang-Feng, T., Tsubokawa. M.,Collins, C., Henzel, W.,

FGF RECEPTOR MULTIGENE FAMILY

41

Le Bon, T., Kathuria, S., Chen, E., Jacobs, S., Francke, U., Ramachandran, J., and Fugita-Yamaguchi, F. (1986). EMBO J. 5,2503-2512. Wagner, J. A., and DAmore, P. A. (1986).J. Cell Biol. 103, 1363-1367. Wahl, M. I., Daniel, T. O., and Carpenter, G. (1988). Science 241, 968-970. Wahl, M. I., Olashaw, N. E., Nishibe, S., Rhee, S. G., Pledger, W. J., and Carpenter, G. (1989). Mol. Cell. Biol. 9, 2934-2943. Walicke, P., Cowan, W. M., Ueno, N., Baird, A., and Guillemin, R. (1986).Proc. Natl. Acad. S C ~U . . S. A. 83, 3012-3016. Wanaka, A., Johnson, E. M., Jr., and Milbrandt, J. (1990). Neuron 5,267-281. Wanaka, A., Milbrandt, J., and Johnson, E. M., J . (1991).Deuelmt(Cambridge, UK) 111, 455-468. Werner, S., Duan, D.3. R., de Vries, C., Peters, K. G., Johnson, D. E., and Williams, L. T. (1992a). Mol. Cell. Biol. 12, 82-88. Werner, S., Peters, K. G., and Williams, L. T. (1992b). In preparation. Williams, A. F., and Barclay, N. A. (1988). Annu. Rev. Immunol. 6 , 381-408. Williams, L. T. (1989). Science 243, 1564-1570. Yarden, Y., Escobedo, J. A., Kuang, W.-J., Yang-Feng, T. L., Daniel, T. O., Tremble, P. M., Chen, E. Y., Ando, M. E., Harkins, R. N., Francke, U., Friend, V. A., Ullrich, A,, and Williams, L. T. (1986). Nature (London) 323, 226-232. Yayon, A., Klagsbrun, M., Esko,J. D., Leder, P., and Ornitz, D. M. (1991). Cell (Cambridge, MOSS.)64,841-848. Zhan, X., Bates, B., Hu, X., and Goldfarb, M. (1988). Mot. Cell. Biol. 8, 3487-3495. Zhu, X., Komiya, H., Chirino, A., Faham, S., Fox, G. M., Arakawa, T., Hsu, B. T., and Rees, D. C. (1991). Science 251, 90-93.